Methods and Systems for Multiplex Gene Amplification from Ultra-Low DNA Input Amounts and Uses Thereof

ABSTRACT

Systems and methods of performing multiplex PCR on low and ultra-low quantities of starting template using custom primer sequences having a homotag. In some embodiments, these primers are capable of amplifying over 100 targets simultaneously and/or are capable of amplifying targets from low quantities of starting template. Along with these primers, sequencing methods are provided capable of sequencing the targets from numerous individuals, simultaneously. Additionally, methods for analyzing the sequencing results to advance treating an individual are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage of PCT Patent Application No. PCT/US2019/050616, entitled “Methods and Systems for Multiplex Gene Amplification from Ultra-Low DNA Input Amounts and Uses Thereof” to Scharfe et al., filed Sep. 11, 2019, which claims priority to U.S. Provisional Application Ser. No. 62/729,921, entitled “Multiplex Gene Sequencing From Ultra-Low DNA Input Amounts” to Scharfe et al., filed Sep. 11, 2018; the disclosure of which are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Governmental support under Grant No. HD081355 awarded by the National Institute of Health. The government has certain rights in the invention.

SEQUENCE LISTING

This application hereby incorporates by reference the material of the electronic Sequence Listing filed concurrently herewith. The material in the electronic Sequence Listing is submitted as a text (.txt) file entitled “06034_Sequences_ST25.txt” created on Aug. 1, 2019, which has a file size of 876 KB, and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to polymerase chain reactions (PCR) and applications thereof, more particularly, multiplex PCR methods that allow for simultaneous amplification of multiple target sequences from ultra-low amounts of template nucleic acids.

BACKGROUND OF THE INVENTION

PCR is a commonly used method in biology and medicine for a number of purposes, including mutation detection, identification of individuals, diagnostic testing, genotyping, and nucleic acid sequencing. Current methods typically can only amplify a limited number of target sequences at a time and require large quantities of high-quality starting template (DNA or RNA). Unfortunately, many biological samples, including dried blood spots, possess low quantities of nucleic acids and can be of limited quality, which makes PCR on these samples difficult and nearly impossible for amplifying multiple targets simultaneously. As such, a need in the art exists to develop systems and methods that enable amplification of multiple target sequences in low quality and/or quantity DNA samples.

SUMMARY OF THE INVENTION

Systems and methods for multiplex nucleic acid amplification in accordance with embodiments of the invention are disclosed. In one embodiment, a composition for performing PCR includes a universal primer and a plurality of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer, wherein the forward and the reverse primer comprises a general 5′-A-B-3′ structure, where A represents the universal primer sequence and B represents a target specific sequence.

In a further embodiment, the universal primer possesses a melting temperature of approximately 69° C. to approximately 72° C.

In another embodiment, the plurality of primer pairs is at least 50 primer pairs.

In a still further embodiment, the plurality of primer pairs is at least 500 primer pairs.

In still another embodiment, the forward primers and reverse primers further comprise a spacer sequence, C, wherein the forward and reverse primers comprise a general 5′-A-C-B-3′ structure.

In a yet further embodiment, the spacer in each forward primer consists of the sequence TCTG and the spacer in each reverse primer consists of the sequence AGAC.

In yet another embodiment, the universal primer and the plurality of primer pairs are at a ratio of 10:1.

In a further embodiment again, the universal primer sequence is SEQ ID: 2818.

In another embodiment again, the target specific sequence for each forward primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 940-1878, and each reverse primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 1879-2817.

In a further additional embodiment, a method of targeted sequencing of an individual, includes the steps of amplifying a plurality of target sequences in a sample using a first PCR reaction to create amplicons containing a universal primer sequence, wherein the first PCR reaction contains a universal primer, a plurality of forward primers, and a plurality of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer, wherein the forward and the reverse primer comprises a general 5′-A-B-3′ structure, where A represents the universal primer sequence and B represents a target specific sequence, generating a sequencing library from the amplicons using a second PCR reaction, wherein the second PCR reaction contains sequencing adapter primers comprising a general 5′-D-A-3′ structure, where D represents a sequencing adapter sequence and A represents the universal primer sequence, and sequencing the sequencing library on a sequencing platform.

In another additional embodiment, the method includes obtaining a sample.

In a still yet further embodiment, the sample is a dried blood spot.

In still yet another embodiment, the forward primers, reverse primers, and sequencing adapter primers further comprise a spacer sequence, C, wherein the forward and reverse primers comprise a general 5′-A-C-B-3′ structure and the sequencing adapter primers comprise a general 5′-D-A-C-3′ structure.

In a still further embodiment again, the universal primer possesses a melting temperature of approximately 69° C. to approximately 72° C.

In still another embodiment again, the universal primer and the plurality of primer pairs are at a ratio of 10:1.

In a still further additional embodiment, the universal primer sequence is SEQ ID: 2818.

In still another additional embodiment, the plurality of primer pairs is at least 50 primer pairs.

In a yet further embodiment again, the plurality of primer pairs is at least 500 primer pairs.

In yet another embodiment again, the target specific sequence for each forward primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 940-1878, and each reverse primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 1879-2817.

In a yet further additional embodiment, the sequencing adapter sequence is selected from the group consisting of SEQ ID NOs: 2819-2820.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings where:

FIG. 1 illustrates a method for obtaining a sample and treating an individual in accordance with embodiments.

FIGS. 2A-2F illustrate primer sequences for a multiplex PCR reaction in accordance with embodiments.

FIGS. 3A-3B illustrate results of multiplex PCR reactions in accordance with embodiments.

FIGS. 4A-4D illustrate primer sequences for a PCR reaction to generate sequencing libraries in accordance with embodiments.

FIGS. 5A-5D illustrate primer sequences for a PCR reaction to generate sequencing libraries in accordance with embodiments.

FIGS. 6A-6C illustrate sequencing reaction primers in accordance with embodiments.

FIGS. 7A-7C illustrate sequencing reaction primers in accordance with embodiments.

FIGS. 8A-8B illustrate results of sequencing reactions in accordance with embodiments.

FIGS. 9A-9C illustrate sequence analysis results in accordance with embodiments.

DETAILED DISCLOSURE OF THE INVENTION

The present disclosure may be understood by reference to the following detailed description, taken in conjunction with the drawings as described below. It is noted that, for purposes of illustrative clarity, certain elements in various drawings may not be drawn to scale.

In accordance with the provided disclosure and drawings, systems and methods of performing multiplex PCR on low and ultra-low quantities of starting template using custom primer sequences having a homotag. In some embodiments, these primers are capable of amplifying over 100 targets simultaneously and/or are capable of amplifying targets from low quantities of starting template. Along with these primers, sequencing methods are provided capable of sequencing the targets from numerous individuals, simultaneously. Additionally, methods for analyzing the sequencing results to advance treating an individual are provided.

Traditional genetic testing or screening typically assesses an individual's genetics via hybridization, PCR, or sequencing. In hybridization panels, an individual's DNA is hybridized to a panel of known variants or mutations to identify which variants the individual possesses. While these panels can typically screen for a large number of targets, the panels are limited in that they can only identify variants that have previously been described and/or identified and cannot identify novel or previously unknown variants and can be limited in the ability to detect structural variation.

Similarly, PCR-based methods are typically limited to known variants but also has a number of problems that arise when amplifying multiple targets within a single reaction, including the increased levels of primers needed for the amplification of each target. With the addition of each target sequence, two additional primers need to be added the reaction. Adding additional primers to a reaction increases the likelihood of forming primer dimers or off-target amplification in the reaction, thus inhibiting amplification of the correct target. One solution has been to add additional template nucleic acid (either DNA or RNA) to the sample to increase the likelihood that the primers will amplify the correct sequence. Another solution is to reduce the concentration of the primers, but this strategy suggests a reduction in PCR sensitivity. Current methods of multiplex PCR have only resulted in amplification of a limited number (e.g., less than about 20) of individual targets in a single reaction. Embodiments herein describe a methods and systems to amplify large numbers of target sequences, including amplifying greater than 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 targets in a single PCR reaction. Further embodiments are directed to methods of sequencing the amplified targets.

Additionally, some biological samples are limited in quantity and/or lack large quantities of DNA or RNA, thus limiting the ability for an individual to amplify multiple targets in a single reaction. For example, dried blood spots (DBSs) are regularly taken from babies shortly after birth. DBSs provide a chance to assess newborns for genetic health defects, disorders, or diseases at a very early time point, which may be important for early life care. However, DBS samples contain only small and varying amounts of blood, thus the nucleic acid content within a DBS is limited. Adding multiple primer pairs to a reaction with limited input template would quickly overwhelm in the input template and increase the likelihood of primer dimers or other inhibiting structures.

Finally, sequencing is a great alternative by providing full sequence reads and identification of novel variants that could be missing from other panels. However, in genetic testing, typically only a panel of genes or genetic elements are relevant, thus whole genome sequencing would reveal much additional data that may not have any effect on underlying diseases or conditions in an individual. Traditional targeted sequencing typically utilizes a combination of hybridization and PCR to isolate and amplify a subset of targets with added costs in reagents, labor, and equipment. Thus, there exists a need in the art for PCR-based panels to amplify a large number of sequences to reduce costs and improve genetic screening, especially with samples containing low amounts of nucleic acids.

An example of a targeted panel of genes is the Recommended Universal Screening Panel (RUSP), which can detect more than forty metabolic disorders that have historically caused significant morbidity and mortality in children. (See American College of Medical Genetics Newborn Screening Expert G. Newborn screening: toward a uniform screening panel and system—executive summary. Pediatrics. 2006; 117(5 Pt 2): 5296-307; and Urv T K, Parisi M A. Newborn Screening: Beyond the Spot. Adv Exp Med Biol. 2017; 1031:323-346; the disclosures of which are incorporated herein by reference in their entireties.) However, typical RUSP assays in newborns uses tandem mass spectrometry (MS/MS). (See Carreiro-Lewandowski E. Newborn screening: an overview. Clin Lab Sci. 2002; 15(4):229-238; Chace D H, Kalas T A, Naylor E W. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clinical chemistry. 2003; 49(11):1797-1817; and Turgeon C, Magera M J, Allard P, et al. Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots. Clinical chemistry. 2008; 54(4):657-664; the disclosures of which are incorporated herein by reference in their entireties.) While beneficial in most respects, MS/MS screening is tuned to maximize the number of newborns identified, with sensitivity favored over specificity. This approach increases the number of false-positive results, leading to considerable emotional and financial burdens of follow-up testing, unneeded medical precautions for false-positive cases and diagnostic delays for some infants. (See Waisbren S E, et al. Effect of expanded newborn screening for biochemical genetic disorders on child outcomes and parental stress. JAMA. 2003; 290(19):2564-2572; the disclosure of which is incorporated herein by reference in its entirety.) To reduce the number of false-positive cases without compromising sensitivity, screen-positive results are followed by second-tier testing at higher specificity. (See Matern D, et al. Reduction of the false-positive rate in newborn screening by implementation of MS/MS-based second-tier tests: the Mayo Clinic experience (2004-2007). Journal of inherited metabolic disease. 2007; 30(4):585-592; the disclosure of which is incorporated herein by reference in its entirety.) As such, second-tier tests measure more specific disease markers (e.g., organic acids) to confirm (true positive) or reject (false positive) the primary screen result. Second-tier tests are typically not part of the primary screen due to assay complexity, limited throughput, analysis time and cost. (See e.g., Chace D H, Hannon W H. Impact of second-tier testing on the effectiveness of newborn screening. Clinical chemistry. 2010; 56(11):1653-1655; the disclosure of which is incorporated herein by reference in its entirety.) However, both primary and secondary screening utilizes the original newborn DBS to avoid a new blood draw and minimize turnaround time.

The advent of rapid, inexpensive next-generation sequencing (NGS) promises to revolutionize newborn screening. (See e.g., Berg J S, et al. Newborn Sequencing in Genomic Medicine and Public Health. Pediatrics. 2017; 139(2); the disclosure of which is incorporated herein by reference in its entirety.) Incorporating NGS-based analysis at the earliest stage in the screening process could drastically streamline the diagnostic work-up following an abnormal NBS result, but has several challenges. Previous studies using residual DBS for NGS either required large amounts of DBS material, or used whole-genome amplification for sequence library preparation. (See Hollegaard M V, et al. Archived neonatal dried blood spot samples can be used for accurate whole genome and exome-targeted next-generation sequencing. Molecular genetics and metabolism. 2013; 110(1-2):65-72; Bhattacharjee A, et al. Development of DNA confirmatory and high-risk diagnostic testing for newborns using targeted next-generation DNA sequencing. Genetics in Medicine: official journal of the American College of Medical Genetics. 2015; 17(5):337-347; Cantarel B L, et al. Analysis of archived residual newborn screening blood spots after whole genome amplification. BMC genomics. 2015; 16:602; and Poulsen J B, et al. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA. PLoS One. 2016; 11(4):e0153253; the disclosures of which are incorporated herein by reference in their entireties.) A less expensive and more efficient approach is multiplex gene sequencing from DBS from a multiplex PCR reaction, using a panel of genes relevant to the specific disease(s) or biological condition(s) detected in primary newborn screening.

Current NGS diagnostics are suboptimal for NBS due to their inability to accommodate DBS-derived material. Newborn DBS samples contain only small and varying amounts of blood, from which multiple punches are taken for NBS for the various conditions on the panel. The small amount of dried blood remaining limits the amount of extractable DNA for use in second-tier testing. Previous studies using residual DBS for NGS either required large amounts of DBS material, or used whole-genome amplification for sequence library preparation. (See Hollegaard M V, et al. Archived neonatal dried blood spot samples can be used for accurate whole genome and exome-targeted next-generation sequencing. Molecular genetics and metabolism. 2013; 110(1-2):65-72; Bhattacharjee A, et al. Development of DNA confirmatory and high-risk diagnostic testing for newborns using targeted next-generation DNA sequencing. Genetics in medicine: official journal of the American College of Medical Genetics. 2015; 17(5):337-347; Cantarel B L, et al. Analysis of archived residual newborn screening blood spots after whole genome amplification. BMC genomics. 2015; 16:602; and Poulsen J B, et al. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA. PLoS One. 2016; 11(4):e0153253; the disclosures of which are incorporated herein by reference in their entireties. A more efficient approach is multiplex gene sequencing from DBS, using a panel of genes relevant to the specific condition(s) detected in primary newborn screening, which is incorporated into numerous embodiments. Further

Turning to FIG. 1, a method 10 is illustrated that incorporates numerous described herein and how many of the embodiments relate into a larger process. In particular, at Step 12, a sample is obtained. In numerous embodiments, the sample contains nucleic acids (e.g., DNA or RNA). In many embodiments, the sample is a blood sample. In some embodiments, the sample is a DBS. It should be noted that many different samples are known in the art.

At Step 14, DNA is isolated from the sample in numerous embodiments. The DNA may be isolated by any means known in the art that is sufficient for the specific tissue and/or source of the sample. Many embodiments will isolate DNA from a DBS using methods designed to yield the maximum quantity of nucleic acids possible. Methods for isolating DNA from DBSs according to many embodiments are described further in depth below.

At Step 16, a PCR reaction is performed in many embodiments. For some embodiments, the PCR reaction amplifies a single amplicon from the template nucleic acids isolated from the sample. In many embodiments, multiplex PCR reactions are performed to isolate many targets simultaneously. Additional embodiments will utilize unique sequences concatenated to target specific primers to increase amplicon efficiency. Additional details on primer design will be described in detail below.

At Step 18 of many embodiments, a sequencing library or target sequences is generated. In a number of embodiments, the sequencing library is generated using PCR. A sequence library in accordance with embodiments will add specific nucleic acid sequences to allow the target amplicons to be sequenced, such as adapter and index sequences. Numerous embodiments will append Illumina adapters to the amplicons generated from the PCR reaction of Step 16.

The library of target sequences will be sequenced at Step 20 of many embodiments. Many methods and platforms for sequencing nucleic acids are known in the art, many of which will be sufficient for sequencing libraries generated in embodiments herein. However, a number of embodiments will utilize an Illumina platform, such as a MiSeq, HiSeq, HiScan, iSeq, MiniSeq, NextSeq, NovaSeq, and/or any other Illumina platform.

Variants will be identified and annotated in the sequence of many embodiments at Step 22 of many embodiments. Numerous methods exist in the art for identifying variants, including GATK, Annovar, and many other available software packages and/or resources.

At Step 24, many embodiments will treat an individual based on the identified and annotated variants. In many embodiments, the treatments are known in the art for an affliction, condition, and/or disease identified at Step 22.

While the above method 10 contains a number of steps, not all steps are necessary to be performed in all embodiments. Additionally, method 10 is meant to illustrate a number of embodiments that stand alone as separate embodiments, which can be integrated into larger processes, methods, systems, kits, etc. Additionally, numerous embodiments may be able to perform some steps simultaneously, nearly simultaneously, and/or in an order that differs from what is illustrated in FIG. 1.

DNA Isolation

Many embodiments are directed to amplifying target sequences using DBS samples collected from individuals, including newborn babies. As noted above, DBS samples contain limited and varying quantities of DNA. As such, many embodiments isolate DNA from DBS in a method to maximize DNA yield. In some embodiments, one or more punches are taken from a DBS. In several embodiments, a single 3 mm punch is taken from a DBS from one individual. In various embodiments, the punch(es) are washed one or more times with 10 mM NaOH. In numerous embodiments, the punch(es) are suspended in a volume of 10 mM NaOH and heated to allow DNA to elute from the DBS. In various embodiments, the punch(es) is suspended in 50 μL of 10 mM. In certain embodiments, the punch(es) are heated for a period of 5, 10, 15, 20, or 30 minutes at 99° C. Various embodiments will mix and/or transfer the liquid, which contains isolated DNA, to a fresh tube for further processing.

Many embodiments will obtain samples from multiple individuals simultaneously. For example, punches can be taken from 96, 192, or 384 individuals simultaneously to allow DNA isolation using 96-well, 192-well, or 384-well plates.

Multiplex PCR Reactions

Many embodiments are directed to components and methods for performing PCR reactions. in accordance with many embodiments is described. Turning to FIGS. 2A-2B, forward 102 and reverse 104 PCR primers in accordance with many embodiments are illustrated. The primers 102, 104 start at the 5′-end 106 to the 3′-end 108 of each primer 102, 104. As with many PCR reactions, forward 110 and reverse 112 target specific primers are included in order to frame the target sequence and allow amplification of the target sequence from template nucleic acids. The amplified target molecules are referred to as amplicons. Additionally, homotags 114, 116 are attached at the 5′-ends 106 of the forward 102 and reverse 104 primers. A homotag in accordance with many embodiments is a universal primer that allows amplification of the amplicons independent of the target sequence. In many embodiments, the homotags 114, 116 that are part of the PCR primers 102, 104 possess the same sequence, thus allowing a single-primer amplification of target amplicons. In general, the structure of primers 102, 104 of many embodiments can be described as 5′-A-B-3′, where A is a homotag 114, 116, and B is target specific primer 110, 112.

In a number of embodiments, the PCR primers 102, 104 will be designed to avoid aberrant amplification, off-target amplification, and/or other issues that may arise because of poor primer design. When designing PCR primers 102, 104, a number of methods can be utilized, including automation with available software packages. In several embodiments, target sequences, such as entire genes, regions, and/or other significant areas, will be analyzed to avoid problematic sequences, such as repetitive elements. Many of these embodiments will utilize repeat masking software, such as RepeatMasker to block off repetitive elements within these regions. For example, if an entire gene sequence is identified as a target sequence, the target sequence may include introns, exons, 5′-UTRs, 3′UTRs, in addition to other genetic elements. Some of these features can include repetitive sequences that can interfere with PCR if used as a target specific primer 110, 112. By masking these sequences, these regions will not be selected as target specific primers 110, 112. Once certain elements are masked, target specific primer 110, 112 will be designed in many embodiments. The primer design can be performed manually or automated using programs such as Primer3. When multiplexing PCR reactions, the target specific primers 110, 112 are designed to have similar characteristics, such as size, melting temperature, GC content, amplicon size of the resulting amplicon, and any combination thereof. In some embodiments, target specific primers 110, 112 will have an average size of approximately 20-30 base pairs (bp), and amplicon size of approximately 300-500 bp. In several embodiments, the target specific primers 110, 112 will range in size from 21-27 bp and have an average size of 23 bp and amplify targets ranging from 350-500 bp with an average size of 412 bp.

Once designed, the entire sequence of PCR primer 102, 104 can be established, including homotags 114, 116. Once fully designed, additional quality control metrics will be performed in a number of embodiments. For example, sequences for PCR primers 102, 104 can be assessed for primer-dimer formation that can interfere with PCR reactions. Methods for optimizing primer design include the AutoDimer software package to assess secondary structure and/or primer dimer formation within a selection of PCR primer. If primers are predicted to form primer dimers or other interfering structures, the target specific primer sequences 110, 112 can be adjusted and reassessed until primer dimers or other structures are minimized.

In many embodiments, a pool of PCR primers 102, 104 are added in a reaction, where the target specific primers 110, 112 differ for the various targets, but the homotags 114, 116 remain the same. In several embodiments, the PCR primers 102, 104 are tested and optimized to reduce amplicon dropout and/or non-specific amplification. This optimization can include rebalancing a pool of primers (raising or lowering the concentration of specific primer pairs) or by altering the characteristics of the target specific primers 110, 112. It should be noted that one of skill in the art is capable of identifying issues with PCR primers, including dropout and/or non-specific amplification, and would know how to rebalance and/or altering primers within a reaction. In a number of embodiments, primer pairs that amplicons with low GC content will be increased, while primer pairs that amplify amplicons with high GC content will be reduced.

In certain embodiments, the homotags 114, 116 are designed to have a different (e.g., higher or lower) melting temperature than the target specific primers 106, 108. By altering melting temperature of the homotags 114, 116, aberrant amplification is less likely to occur from the presence of the homotags 114, 116. Additional embodiments will design homotags 114, 116 to lack homology with sequences within the genome sequence of a sample to be amplified. Lacking homology with the sample's genome will aid in preventing aberrant or erroneous amplification. Many methods exist for determining which sequences possess or lack homology, including performing alignments of a particular sequence to the sample's reference genome sequence (e.g., using BLAT, BLAST, and/or any other alignment software) or querying a K-mer database. It should also be understood that lacking homology does necessarily not mean possessing no homology with a reference sequence but lacking sufficient homology to prevent amplification under particular PCR reaction conditions.

In numerous embodiments, the homotags 114, 116 will have a higher melting temperature. Having a higher melting temperature for the homotags 114, 116 will allow for amplification of all amplicons using homotag-specific primers without allowing the target specific primers 110, 112 to anneal to template nucleic acids. In such a circumstance, amplicon amplification will occur rather than template amplification—i.e., amplification will be based on amplicons containing homotag sequences rather than generating new amplicons from sample template. It should be noted that because nucleic acid amplification is directional from 5′ to 3′, homotag-specific primers will have the same sequence as the homotags 114, 116, such as illustrated in FIGS. 2E-2F, where the homotag-specific primers are only the homotag sequences 114, 116.

Turning to FIGS. 2C-2D, a number of embodiments will include forward 118 and reverse 120 spacer sequences between homotags 114, 116 and target specific primers 110, 112. Spacer sequences 118, 120 can be used to provide directionality into PCR primers 102′, 104′. Spacer sequences 118, 120 can be of any length to allow differentiation in direction. In a number of embodiments, the spacer sequences are 4-8 bp long. In certain embodiments, the spacer sequences are 4 bp sequences. In embodiments including spacers, the primers 102′, 104′ can be described to have a general structure of 5′-A-C-B-3′, where A is a homotag 114, 116, B is a target specific primer 110, 112, and C is a spacer 118, 120.

The RUSP panel contains 60 conditions including 34 core conditions and 26 secondary conditions. In a number of embodiments, the targets are selected based on the RUSP panel. In some embodiments, a panel of 72 genes are selected that include 64 genes associated with 46 different RUSP metabolic disorders and cystic fibrosis and an additional 8 genes associated with 7 metabolic disorders that are not currently in the RUSP metabolic disorders. Table 1 provides a list of conditions selected in some embodiments for use in many embodiments. The list in Table 1 includes conditions currently in the RUSP panel as well as additional conditions that will be selected in certain embodiments. In particular Table 1 identifies specific conditions in a RUSP panel by its ACMG code, the specific condition identified by that code, whether it is a core or secondary condition (if the condition is in the RUSP panel), condition type, genes and NCBI numbers for those genes that are associated with the condition, the current methodologies for determining the condition and the primary analyte for the analysis. Additional information regarding metabolic conditions can be found at www.hrsa.gov/advisory-committees/heritable-disorders/rusp/index. html; the disclosure of which is incorporated by reference in its entirety.

In a number of embodiments targeting the 72 genes as described in Table 2, the specific segments of these genes are selected as target amplicons. In many embodiments, the target amplicons are selected as all exons, flanking intronic regions, and key non-coding regions. In certain embodiments, the targeted amplicons are selected from the group consisting of SEQ ID NOs: 1-939. In a number of embodiments, forward target specific primers 110 are selected from SEQ ID NOs: 940-1878 and reverse target specific primers 112 are selected from SEQ ID NOs: 1879-2817. Table 2 lists specific target names and identifies SEQ ID NOs for the target sequence and correlating forward and reverse target specific primers, where the forward primer SEQ ID NO and the reverse primer SEQ ID NO in a row form a primer pair for the target sequence SEQ ID NO in that same row. For example, SEQ ID NO: 940 and SEQ ID NO: 1879 form a primer pair for SEQ ID NO: 1. It should be noted that the specific sequence for any of the target sequences SEQ ID NOs: 1-939 are only representative of the specific target to be amplified. In many embodiments, variants will exist within the target amplicon for a particular sample, thus one primer pair will amplify a target sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a particular target sequence.

In certain embodiments, the homotag is SEQ ID NO: 2818. In some embodiments including spacers 118 and/or 120, the spacers are selected from the 5′-TCTG-3′ and 5′-AGAC-3′. In several embodiments of directional PCR primers 102′, 104′ (e.g., primers including spacers 118, 120) the forward spacer is 5′-TCTG-3′, and the reverse spacer is 5′-AGAC-3′. Thus, In many embodiments, the forward PCR primer 102 has a general structure of 5′-X-Y-Z-3′, where X is SEQ ID NO: 2818, Y is TCTG, and Z is any one of SEQ ID NOs: 940-1878, and reverse PCR primer 104 has a general structure of 5′-X-Q-R-3′, where X is SEQ ID NO: 2818, Q is AGAC, and R is any one of SEQ ID NOs: 1879-2817. Numerous embodiments will pool multiple versions of PCR primers 102 and 104 or directional PCR primers 102′ and 104′.

An advantage having homotags 114, 116 with higher melting temperatures is that it will allow for a single reaction set up, which includes PCR primers 102, 104 or directional PCR primers 102′, 104′ along with homotag-specific primers 114, 116 within the same reaction tube or vessel. In such a circumstance, the reaction will comprise template nucleic acid (e.g., DNA and/or RNA), buffer, water, one or more forward primers, one or more reverse primer, a homotag-specific primer, nucleotide triphosphates (e.g., dNTPs and/or NTPs), a polymerase, and/or any other component known in the art to assist or promote PCR amplification. In many embodiments the pool PCR primers 102, 104 or directional PCR primers 102′, 104′ will be used at a total concentration of approximately 0.5 μM (e.g., ±0.5 μM), such that some embodiments will utilize 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1.0 μM of total PCR primers 102, 104 or directional PCR primers 102′, 104′. The homotag-specific primer 114, 116 will be placed in the reaction tube at a concentration equal to or greater than the concentration of pooled PCR primers 102, 104 or directional PCR primers 102′, 104′. As such, many embodiments will use a ratio of homotag-specific primers to PCR primers of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or greater for the ratio of homotag-specific primers to PCR primers. Many embodiments will use a ratio of 10:1 homotag-specific primers to PCR primers, such that approximately 5 μM of homotag specific primers 114, 116 will be used in a reaction containing approximately 0.5 μM of pooled PCR primers 102, 104 or directional PCR primers 102′, 104′.

Many embodiments are directed to DNA polymerization based on a DNA template, so these reactions will comprise a DNA template, buffer, at least one forward primer, at least one reverse primer, a homotag primer, dNTPs, and a polymerase. In many embodiments, the forward and reverse primers will possess a homotag sequence, such as those described herein. In many embodiments, the polymerase will be a DNA polymerase, such as Taq polymerase, while additional embodiments will utilize high fidelity polymerases, strand displacement polymerases, RNA polymerases, long-range polymerases, any other polymerase relevant to the type of reaction, and/or any combination thereof. Numerous embodiments will alter the temperature cycling of the reaction to include a first set of cycles with a lower annealing temperature to allow for template amplification followed by a second set of cycles with a higher annealing temperature to allow for amplicon amplification. For example, the first set of cycles will have an annealing temperature of approximately 50-69° C. followed by a second set of cycles with an annealing temperature of approximately 70-72° C. Additional embodiments will include manipulations to the sets of cycles, such as ramping, touch-down, or any other methodology for amplification. Some specific embodiments utilize the following profile:

-   -   95° C. for 3 minutes,     -   12 cycles of:         -   95° C. for 16 seconds,         -   69-52° C. for 2 minutes (reducing temperature by 1.5° C. per             cycle),         -   72° C. for 45 seconds,     -   10 cycles of:         -   95° C. for 16 seconds,         -   72° C. for 20 seconds,         -   72° C. for 2 minutes.

While the above is described in relation to a single sample, many embodiments will utilize common reaction plates to allow PCR amplification from multiple samples obtained from multiple individuals simultaneously. For example, 96 individual samples can be kept in a standard 96-well plate, which would allow for multiplex PCR reactions to be performed on all 96 samples simultaneously.

Turning to FIGS. 3A-3B, the success rates of amplifying 939 amplicons are illustrated in accordance with embodiments. Specifically, FIG. 3A illustrates the number of sequencing reads per sample, which indicates the overall success rate of numerous embodiments of multiplex PCR reactions. As seen in FIG. 3A, many samples were capable of producing approximately 1 million reads (e.g., 10⁶ reads). As illustrated, out of 78 samples, only 1 sample produced significantly fewer reads than other samples. Additionally, FIG. 3B illustrates partial failure of multiplex PCR reactions as a function of uniformity, in accordance with many embodiments using DBS as the sample source. In particular, FIG. 3B illustrates the percent of amplicons with at least 20% of the mean coverage of all amplicons. As illustrated in FIG. 3B, only 2 samples out of 78 showed limited uniformity among the amplicons. In combination, FIGS. 3A-3B illustrate that many embodiments are capable of amplifying upwards of 900 amplicons from a very low amount of input nucleic acids.

Generating Sequencing Libraries

Many sequencing library generation methods are known in the art, including commercially prepared kits for building such libraries, such as those from KAPA, Illumina, and other companies. However, many of these kits rely on ligating adapters to the ends of the target molecules rather than purely through PCR. Many embodiments will leverage the power of the homotags or homotags and spacer sequences to generate sequencing libraries for the target sequences.

Upon amplifying target sequences, a number of embodiments will generate sequencing libraries from the amplicons created during a PCR reaction, such as a PCR reaction described above. In certain embodiments, the sequencing libraries are created using a second PCR reaction. In a second reaction, additional primers can utilize the homotags to add additional adapters necessary for sequencing (e.g., IIlumina P5 and/or P7 adapters). However, using the homotag sequences alone may not provide adequate representation of all amplicons in a reaction. For example, Illumina sequencing relies on different adapters residing at each end of a molecule to be sequenced. Using only the homotag sequences as primers would create an equal opportunity for the directional Illumina adapters to be added to each end, thus resulting in 50% of all molecules having the same sequencing adapter at each end of the molecule (P5-P5 or P7-P7). As such, many embodiments will utilize spacer sequences to create directionality for adding the sequencing adapters to the amplicons. As such, many adapters will have a structure as illustrated in FIGS. 4A-4B. In FIGS. 4A-4B, sequencing adapter primers 122, 124 utilize the homotag 114 and spacer sequences 118, 120 to amplify the amplicons. By using forward 118 and reverse 120 spacer sequences, attached at the 3′-end 108 of the sequencing adapter primers 122, 124, the system can allow for only one sequencing adapter 126, 128 is added to each end of a molecule. In general, sequencing specific primers 122, 124 can be described to have the structure 5′-D-A-C-3′, where D is a sequencing adapter 126, 128, A is a homotag 114, 116, and C is a spacer sequence 118, 120. In many embodiments, the Illumina P5 adapter (SEQ ID NO: 2819) and Illumina P7 adapter (SEQ ID NO: 2820) will be used for the sequencing adapters 126, 128. In some embodiments, the forward sequencing adapter primer 122 will possess the structure 5′-Illumina P5 (SEQ ID NO: 2819)—homotag (SEQ ID NO: 2818)—forward spacer (TCTG)-3′, and the reverse sequencing adapter primer 124 will possess the structure 5′-Illumina P7 (SEQ ID NO: 2820)—homotag (SEQ ID NO: 2818)—reverse spacer (AGAC)-3′.

Further embodiments of sequencing adapter primers will include index sequences to allow for multiplex sequencing of multiple samples simultaneously. Many methods are known in the art to index and/or multiplex samples for sequencing. In a number of embodiments, a Nextera-style indexing system is used. Nextera indexing is a system that integrates one or two indexes onto the molecules in a sequencing library. By using two indexes, a specific combination of indexes identifies a single sample. For example, a set of 8 indexes at a first location and a set of 12 indexes at a second location creates 96 unique combinations, thus allowing a total of 20 indexes to uniquely identify 96 individual samples, which can be used for embodiments that have performed PCR reactions on 96 individual samples. Many embodiments will utilize index sequences, as shown in Table 3:

TABLE 3 List of Indexes First Indexes Second Indexes 5′-TAGATCGC-3′ 5′-TCGCCTTA-3′ 5′-CTCTCTAT-3′ 5′-CTAGTACG-3′ 5′-TATCCTCT-3′ 5′-TTCTGCCT-3′ 5′-AGAGTAGA-3′ 5′-GCTCAGGA-3′ 5′-GTAAGGAG-3′ 5′-AGGAGTCC-3′ 5′-ACTGCATA-3′ 5′-CATGCCTA-3′ 5′-AAGGAGTA-3′ 5′-GTAGAGAG-3′ 5′-CTAAGCCT-3′ 5′-CCTCTCTG-3′ 5′-AGCGTAGC-3′ 5′-CAGCCTCG-3′ 5′-TGCCTCTT-3′ 5′-TCCTCTAC-3′

In many embodiments, the indexes shown in Table 3 are integrated between sequencing adapters and the homotags, such as illustrated in FIGS. 4C-4D. In FIGS. 4C-4D, sequencing adapter primers 122′, 124′ are illustrated. Specifically, FIG. 4C illustrates a first index 130 integrated between sequencing adapters 1 126 and homotag 114, while FIG. 4D illustrates a second index 132 integrated between sequencing adapter 2 128 and homotag 114. In general, sequencing specific primers 122′, 124′ can be described to have the structure 5′-D-E-A-C-3′, where D is a sequencing adapter 126, 128, E is an index sequence 130, 132, A is a homotag 114, 116, and C is a spacer sequence 118, 120. As such, in some embodiments, the forward sequencing adapter primer 122′ will possess the structure 5′-Illumina P5 (SEQ ID NO: 2819)—first index (Table 3)—homotag (SEQ ID NO: 2818)—forward spacer (TCTG), and the reverse sequencing adapter primer 124′ will possess the structure 5′-Illumina P7 (SEQ ID NO: 2820)—second index (Table 3)—homotag (SEQ ID NO: 2818)—reverse spacer (AGAC)-3′.

However, many embodiments will utilize custom sequencing read primers based on the homotags 114, 116; in some of these embodiments customs sequencing read primers will incorporate homotags 114, 116 and spacers 118, 120. In such embodiments, additional base pairs may be necessary to raise the melting temperature of the sequencing read primers. FIG. 5A illustrates additional sequencing adapter primers 122″, 124″ that include modifying spacers 134, 136 between sequencing adapters 126, 128 and homotags 114, 116. Additionally, FIG. 5B illustrates sequencing adapter primers 122′″, 124′″ that include modifying spacers 134, 136 between indexes 130, 132 and homotags 114, 116. In general, the sequencing adapter primers 122″, 124″ can be described to have the structure 5′-D-F-A-C-3′, while sequencing adapter primers 122′″, 124′″ can be described to have the structure 5′-D-E-F-A-C-3′. In these embodiments, D is a sequencing adapter 126, 128, E is an index sequence 130, 132, F is a modifying spacer 134, 136, A is a homotag 114, 116, and C is a spacer sequence 118, 120.

In many embodiments, the first modifying spacer 134 will be a dinucleotide GG increase the melting temperature of a first sequencing read primer. Additional embodiments will utilize the oligonucleotide CCGTTTA as the second modifying spacer 136 to increase the melting temperature of a second sequencing read primer. As such, some embodiments of the forward sequencing adapter 122″ will possess the structure 5′-Illumina P5 (SEQ ID NO: 2819)—first modifying spacer (GG)-homotag (SEQ ID NO: 2818)—forward spacer (TCTG)-3′, and the forward sequencing adapter primer 122′″ will possess the structure 5′-Illumina P5 (SEQ ID NO: 2819)—first index(Table 3)-first modifying spacer (GG)-homotag (SEQ ID NO: 2818)—forward spacer (TCTG)-3′. Similarly, some embodiments of the reverse sequencing adapter primer 124″ will possess the structure 5′-Illumina P7 (SEQ ID NO: 2820)—second modifying spacer (CCGTTTA)-homotag (SEQ ID NO: 2818)—reverse spacer (AGAC)-3′, and reverse sequencing adapter primer 124′″ will possess the structure 5′-Illumina P7 (SEQ ID NO: 2820)—second index (Table 3)—second modifying spacer (CCGTTTA)-homotag (SEQ ID NO: 2818)—reverse spacer (AGAC)-3′.

Many possible PCR cycling conditions can be used to create the sequencing libraries, based on the enzymes used, the melting temperature of the primers, and the length of the target molecules. In several embodiments the following cycling conditions are used:

-   -   98° C. for 16 seconds,     -   13 cycles of:         -   98° C. for 16 seconds,         -   72° C. for 20 seconds.

In a number of embodiments, the sequencing libraries are cleaned and/or purified after generation. In some embodiments, the cleaning uses commercially available kits, including kits using beads and/or columns. Some embodiments will use AMPure XP beads with a beat to sample ratio of 0.65:1, after which the sequencing libraries are eluted in 50 μL of water.

Further embodiments will size select the library to eliminate too long or too short fragments, which could be generated from primer dimers and/or off target amplification. Many kits exist to perform such size selection, which can be used in embodiments. Some embodiments will utilize a Pippin Prep system for size selection.

Additional embodiments will also quantify the library, which can be accomplished with many means, including UV-Vis spectroscopy, fluorescence, qPCR, and/or electrophoresis. Certain embodiments will perform library quantification using a Bioanalyzer.

Sequencing Targets

As noted above, many possible sequencing platforms can be utilized to sequence targets generated in many embodiments. Also, many embodiments will utilize an Illumina platform to sequence the targets, including an Illumina MiSeq. Sequencing can be performed in any capacity allowed by a particular piece of equipment, including single read, paired-end reads, and/or mate-pair reads. Many embodiments will utilize paired-end read capacity of the platform in order to obtain as much sequence as possible for a particular target, including the entirety of the target sequence.

With such a configuration as illustrated in FIGS. 6A-6C, custom sequencing read primers can utilize the homotags and spacer sequences as custom sequencing read primers. Specifically, FIG. 6A illustrates a first read sequencing primer 138; FIG. 6B illustrates a second read sequencing primer 140 in accordance with many embodiments; and FIG. 6C illustrates an indexing read primer 142, for embodiments including an index. In these embodiments, the sequencing read primers 138, 140 comprise a homotag 114, 116 located at the 5′-end of a spacer sequence 118, 120. In this configuration, the sequencing primers 138, 140 assure directionality of sequencing reads. In general, the sequencing read primers can be described to have the structure 5′-A-C-3′, where A is a homotag 114, 116, and C is a spacer sequence 118, 120. In embodiments which include Nextera-style indexes, a separate indexing read is necessary to read a second index. In these embodiments, the indexing read primer 142 is typically a reverse complement of second read sequencing primer 140. This configuration is illustrated in FIG. 6C, which illustrates a reverse complement of reverse spacer 120′ at the 5′-end of reverse complement of homotag 116′. Thus, in general, indexing read primer 142 can be described to have the structure 5′-C′-A′-3′, where C′ is the reverse complement of a reverse spacer 120′, and A′ is the reverse complement of a homotag 116′. In many embodiments, first sequencing read primer 138 will have the sequence of SEQ ID NO: 2821, which represents the structure 5′-homotag (SEQ ID NO: 2818)—forward spacer (TCTG)-3′, and second sequencing read primer 140 will have the sequence of SEQ ID NO: 2822, which represents the structure 5′-homotag (SEQ ID NO: 2818)—reverse spacer (AGAC)-3′. Also, when an indexing read primer 142 is used, it will have the sequence of SEQ ID NO: 2823, which represents the reverse complement of 5′-homotag (SEQ ID NO: 2818)—reverse spacer (AGAC)-3′.

As noted above, some embodiments will include modifying spacers 136, 138 to increase the melting temperature of the sequencing primers. FIGS. 7A-7C illustrate embodiments which include modifying spacers 136, 138 to increase the melting temperature. In particular, FIGS. 7A-7B illustrate sequencing read primers 138′, 140′ comprising a modifying spacer 136, 138 located at the 5′-end of a homotag 114, 116, which is located at the 5′-end of a spacer sequence 118, 120. In this configuration, the sequencing primers 138, 140 assure directionality of sequencing reads and an increased melting temperature to comply with machine settings, uniformity, or any other relevant factor for ensuring proper sequencing reads. In general, the sequencing read primers can be described to have the structure 5′-F-A-C-3′, where F is a modifying spacer 136, 138, A is a homotag 114, 116, and C is a spacer sequence 118, 120. In embodiments which include Nextera-style indexes, a separate indexing read is necessary to read a second index. In these embodiments, the indexing read primer 142′ is typically a reverse complement of second read sequencing primer 140′. This configuration is illustrated in FIG. 7C, which illustrates a reverse complement of reverse spacer 120′ at the 5′-end of reverse complement of homotag 116′, which is located at the 5′-end of reverse complement of a second modifying spacer. Thus, in general, indexing read primer 142′ can be described to have the structure 5′-C′-A′-F′-3′, where C′ is the reverse complement of a reverse spacer 120′, A′ is the reverse complement of a homotag 116′, and F′ is the reverse complement of a second modifying spacer 136′. In many embodiments, first sequencing read primer 138′ will have the sequence of SEQ ID NO: 2824, which represents the structure 5′-first modifying spacer (GG)-homotag (SEQ ID NO: 2818)—forward spacer (TCTG)-3′, and second sequencing read primer 140′ will have the sequence of SEQ ID NO: 2825, which represents the structure 5′-second modifying spacer (CCGTTTA)-homotag (SEQ ID NO: 2818)—reverse spacer (AGAC)-3′. Also, when an indexing read primer 142′ is used, it will have the sequence of SEQ ID NO: 2826, which represents the reverse complement of 5′-second modifying spacer (CCGTTTA)-homotag (SEQ ID NO: 2818)—reverse spacer (AGAC)-3′.

The raw data from a sequencer can be handled with innate software within the sequencing platform to generate the sequence files, including de-multiplexed sequence files (where multiple samples were multiplexed in the sequencing run). For example, MiSeq Control Software and MiSeq Reporter can analyze the raw image data and de-multiplexing of a run during and/or after a sequencing run has come to completion. Many embodiments will output the sequence in FASTA and/or FASTQ files for further analysis.

Turning to FIGS. 8A-8B, the success of sequencing libraries generated from embodiments are illustrated. In particular, FIG. 8A illustrates that embodiments produce consistent read depths across multiple sequencing runs. Additionally, FIG. 8B illustrates the success in sequencing individual samples, where the percent of base pairs with greater than 20× coverage are illustrated. As seen in FIG. 8B, most samples tested produced at least 20× coverage in greater than 90% of all bases sequenced, and only 1 sample (out of 78 samples) produced lower read depth.

Identify and Annotate Variants

Once sequences have been generated, as above, the target sequences can be analyzed to identify variants and/or possible genetic conditions associated with these variants. In many embodiments, the sequences for each sample are aligned to a reference genome to identify particular variants. Alignment can be performed using any known software package for performing such alignments, including BLAST, BLAT, BWA, among others. Once sequencing reads are aligned, certain embodiments will identify variants using known software packages, including GATK or similar software packages. Variants in accordance with many embodiments including single nucleotide variants (SNVs), copy number variants (CNVs), and insertion-deletion variants (indels). Once variants are identified, a number of embodiments will annotate the variants for nomenclature and/or disease associations using databases of such information, including HGVS, OMIM, dbSNP, ClinVar, and ExAC. An example of such output can be seen in Table 4. Table 4 illustrates results from an embodiment identifying a sample ID (e.g., specific coordinate on sample plate), underlying genetic condition, and numerous categories identifying genes with multiple pathologic (P) and/or likely pathologic (LP) variants, genes with variants of unknown significance (VUS), and PubMed identifiers for known variants identified. Many embodiments will automate this process by pipelining all analysis beginning from the sequence reads to an output of relevant annotations for an individual.

FIGS. 9A-9C summarize variants identified in many embodiments. In particular, FIG. 9A shows the distribution of the number of variants identified in control, false positive (MMA.FP) and true positive (MMA.TP) samples across 72 genes utilized in many embodiments. Similarly, FIG. 9B illustrates the number of variants distribution of the number of variants identified in control, false positive (as identified via MS/MS) (MMA.FP) and true positive (MMA.TP) samples across 8 MMA genes utilized in some embodiments. FIG. 9C illustrates the improvement in numerous embodiments, where pathogenic or likely pathogenic (P/LP) variants were identified in the true positive samples, thus reducing false positive rates, which reducing the demand for second-tier screening for many individuals, which in turn reduces the financial and/or emotional burden of receiving false positive results.

Treatment

In many embodiments, the results of sequencing and/or analysis, such as those described above will guide treatment of an individual. In certain embodiments, the results of the sequencing and/or analysis will be provided to a treating medical provider, such as a physician, nurse, or any other medical professional capable of providing treatment. Upon receiving such information as to metabolic conditions or other genetic diseases, the medical professional can utilize the information to select a treatment and provide the treatment to the individual. In many embodiments, the treatment step is an intervention, such as a drug, device, surgery, or other treatment designed to obviate symptoms and/or indications of the disease or condition, while additional embodiments will provide prophylaxis to the individual to prevent the onset of symptoms and/or complications that can arise due to the presence of a particular disease or condition identified from the sequencing and/or analysis.

EXEMPLARY EMBODIMENTS

Sequencing data supports the notion that embodiments described herein are capable of high plexity PCR amplification of target amplicons from very low nucleic acid inputs. The following data also details the ability to identify numerous metabolic conditions based on the presence of variants identified from target amplification. Accordingly, these data support the various embodiments of the invention as described.

Example 1: Amplifying and Sequencing Optimizing Sulfhydryl Blocking to Produce EVs

Background: DBS samples contain small and varying amounts of blood, thus contain very limited amounts of nucleic acids, including DNA. As such, analysis of metabolic diseases, through biochemical or chemical techniques or through genetic analysis is difficult.

Methods: In one exemplary embodiment, multiplex PCR and sequencing were performed for 939 amplicons starting from 80 DBS samples.

Study specimens: Research was approved by the Institutional Review Boards at Yale University (Protocol ID: 1505015917), Stanford University (Protocol ID: 30618) and the State of California Committee for the Protection of Human Subjects (Protocol ID: 13-05-1236). De-identified residual DBS samples from 80 newborns from the California Biobank Program were used to validate the assay of this embodiment. These samples included 30 confirmed MMA cases, 30 MMA screen false-positives, and 20 DBS from healthy controls. In addition, metabolic data from a larger cohort of 803 newborns, consisting of 103 cases with confirmed MMA (24 mut0, 26 mut-, 45 Cbl C, D, or F, 3 Cbl A or B, and 5 unclassified MMA), 502 screen false-positives, and 198 healthy controls were evaluated. All newborns had routine MS/MS metabolic screening performed through the California NBS program between 2005 and 2015. The 56 MS/MS analytes included free carnitine, acylcarnitines, amino acids and calculated ratios. Additional data collected included newborn race/ethnicity, gestational age (GA, in days), birth weight (in grams), total parenteral nutrition (yes or no), and newborn age at blood collection (in hours).

DNA Extraction: A single 3 mm punch was taken from each DBS using a PE Wallac instrument (Perkin Elmer, Santa Clara) and deposited into a 96-well plate. Three blank paper spots were punched between each sample to prevent cross-contamination. DBS punch spots were washed twice with 180 μL of 10 mM NaOH. Each punch spot was then suspended in 50 μL of 10 mM NaOH solution and heated at 99° C. for 15 minutes in an Applied Biosystems GeneAmp PCR System 9700 (Life Technologies, Grand Island, N.Y.). The supernatant, containing eluted DNA, was mixed by pipetting and then transferred to a clean tube containing 50 μL of 20 mM TrisCL pH 7.5. Two samples (D3, C11 in Table 4) of the 80 DBS failed in the DNA extraction.

Primer Design: A custom script integrating the primer design code from Primer 3 was used to generate target-specific forward and reverse primers for 939 amplicons for 362,013 base pairs (bp) of all exons and 20 bp of flanking intronic sequence of 72 genes based on hg19/GRCh37 (Table 2). Primer hybridization sites were selected to avoid common variants found in the National Center for Biotechnology Information (NCBI) single nucleotide polymorphism Database (dbSNP) build 137, June 2012 release. Primers were designed to have similar length (average 23 bp; range 21-27 bp), GC content, and amplicon size (average 412 bp, range 350-450 bp), matching the 2×250 bp paired-end sequencing chemistry on the MiSeq instrument (Illumina, San Diego, Calif.). Exons larger than 350 bp were covered by overlapping amplicons. Adapter sequences (e.g., homotag sequence SEQ ID NO: 2818) (24 bp) were included at the 5′ end of each primer (e.g., SEQ ID NOs: 940-2817) for post-capture amplification.

Multiplex Target Capture: The 939 primer pairs (e.g., primers consisting SEQ ID NO: 2818 coupled to each of SEQ ID NOs: 940-2817) were pooled in one (1) tube for multiplex amplification 5 of 72 genes. Establishing a multiplex reaction in this embodiment required careful primer design and primer pool rebalancing, that included increasing or lowering the concentration of specific primers, replacing of failed primers, sequencing and data analysis. Primer optimization minimized amplicon dropout and non-specific amplification and achieved a 99% target base coverage from <10 ng of DBS DNA. Multiplex PCR was performed in a Veriti 96-well thermal cycler (Applied Biosystem, Foster City, Calif.) using 4-6 μL of extracted DNA in a 20 μL final volume and the KAPA2G Fast Multiplex PCR Kit (Kapa Biosystems, Wilmington, Mass.) across the following thermal profile: 95° C. for 3 minutes, 12 cycles of 95° C. for 16 seconds, 69-52° C. (−1.5° C. per cycle) for 2 minutes, and 72° C. for 45 seconds, followed by 10 cycles of 95° C. for 16 seconds, 72° C. for 20 seconds, and 72° C. for 2 minutes. PCR cleanup was performed by adding 14 μL (0.7:1) of AMPure XP beads (Beckman Coulter, Brea, Calif.) and clean up according to the manufacturer's manual, with a final elution in 14 μL elution buffer.

Sequence Library Construction: 78 samples in four MiSeq runs were sequenced by multiplexing 17 to 22 samples per run. A no-template water control was included in each run. Sequencing library preparation was performed according to the manufacturer's instructions (Illumina, San Diego, Calif.) using 5 μL of PCR product per sample. PCR was set up in 25 μL reactions, using common primers with sample specific indices and Illumina's P5 (SEQ ID NO: 2819) and P7 (SEQ ID NO: 2820) adapter sequences attached at the 5′ end. Samples were barcoded with 8 bp dual indices (Table 3) according to Illumina's index sequencing protocol. KAPA2G Fast Multiplex PCR Kit (Kapa Biosystems, Wilmington, Mass.) was used to amplify DNA samples with the following cycling conditions: 98° C. for 16 seconds, 13 cycles of 98° C. for 16 seconds and 72° C. for 20 seconds. Following DNA quantification for each sample, samples were pooled (approximately 200-400 μL total volume) and purified using AMPure XP beads 5 (Beckman Coulter, Brea, Calif.) with a bead to sample ratio of 0.65:1 and eluted in 50 μL. 30 μL of the eluate was used for fragment size selection (440-720 bp) using the Pippin Prep system (Sage Science Inc., Beverly, Mass.), quantified the NGS library using Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif.), and performed 2×250 bp PE sequencing on MiSeq (Illumina, San Diego, Calif.).

Sequence Data Analysis: Image analysis and sample de-multiplexing was performed with the Illumina MiSeq Control Software version 2.4.1 and MiSeq Reporter version 2.5.1.3 (Illumina, San Diego, Calif.). The resulting processed fastq files were aligned to the GRCh38 human reference genome using the Burrows-Wheeler Aligner (BWA-MEM, version 0.7.13-r1126). Picard (version 2.8.1) was used to sort and convert files to BAM format. Quality control (QC) metrics were extracted for each sample from the BAM file, including total number of reads, percent reads that were properly paired and mapped to the reference genome, read depths for each amplicon, and read depth for individual base pairs within the target region (FIGS. 3A-3B and FIGS. 8A-8B). DNA variant calling was performed using GATK (version 3.6-0-g8967209) with parameters as described previously. (See e.g., Lefterova M I, et al. Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis. J Mol Diagn. 2016; 18(2):267-282; the disclosure of which is incorporated herein by reference in its entirety.) Annovar was used to annotate variants with the corresponding HGVS DNA and protein level nomenclature in combination with public information relevant for variant annotation from OMIM, dbSNP, ClinVar and ExAC. For each sample (Table 3), sequence variants were classified as pathologic (P), likely pathologic (LP), likely benign, benign, or of unknown significance (VUS) based on ACMG standards and guidelines for interpretation of sequence variants.

Results: This embodiment shows the development of a highly multiplex PCR and NGS method for the analysis of 939 amplicons derived from 72 genes for inborn metabolic disorders from DBS. Out of 80 starting samples, only 2 samples failed the DNA extraction protocol. FIG. 3A illustrates that only 1 of the remaining 78 samples failed in the multiplex PCR reaction, indicating the protocol is robust. Additionally, FIG. 3B illustrates that only 2 samples had partially failed amplification as indicated by 2 standard deviations below the mean depth coverage of all amplicons, one of these samples also failed the total multiplex PCR amplification. Additionally, FIG. 8A illustrates the robustness of embodiments, with consistent read depth across 4 sequencing runs on an Illumina MiSeq. Finally, FIG. 8B illustrates the per-base coverage in all samples. Again, only one sample had significantly lower depth coverage, which is the same sample that failed total and partial amplification. In the end, 77 out of 80 DBS samples were able to proceed to full sequence analysis with greater than 90% of all bases having at least 20× coverage, which provides high confidence for variant calling.

Table 4 summarizes many of the results for the 77 sequenced samples that were successfully sequenced. These 77 samples include 28 MMA patients, of which 25 patients were identified with two variants in an MMA gene, while two patients (B2 and F4) had only one P/LP variant and one patient (F3) had no variant in the eight MMA genes analyzed. In the 29 MMA false-positive cases, two samples (E10 and H10) were detected with two variants in an MMA gene, which in both samples were found in cis on the same amplicon reads and are thus located on the same chromosome. In the 20 control samples, two variants in an MMA gene were not detected. Analysis of the other 64 genes in this embodiment identified samples with two P/LP variants in PAH, PCCA, MTHFR, MLYCD, HPD, ACADVL, FAH, CPS1, DBT, and NAGS. In two 15 samples (B8 and H6) the two P/LP were found on the same chromosome in MLYCD and PAH, respectively.

Conclusion: The results illustrate the ability to amplify very high amounts of amplicons from an ultra-low amount of starting DNA. From the multiplex PCR reaction, sequencing libraries can be constructed to identify variants and underlying metabolic and/or genetic conditions for an individual based on the low levels of starting DNA. While 2 samples failed the DNA extraction protocol, only 1 sample failed the amplification and sequencing reactions, emphasizing the power and robustness of embodiments.

DOCTRINE OF EQUIVALENTS

While the above description contains many specific embodiments of the invention, these should not be construed as limitations on the scope of the invention, but rather as an example of one embodiment thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their equivalents.

TABLE 1 Curation of 72 metabolic genes Recommended Uniform Screening Panel (RUSP)* Condition NCBI NBS Primary ACMG code Conditions Category type Gene Gene method analyte PROP Propionic Acidemia core Metab-OA PCCA 5095 DBS, C3 PCCB 5096 MS/MS MUT Methylmalonic Acidemia (mut-0, mut-) core Metab-OA MUT 4594 DBS, C3 MS/MS Cbl-A, B Methylmalonic Acidemia (Cobalamin disorders) core Metab-OA MMAA 166785 DBS, C3 MMAB 326625 MS/MS IVA Isovaleric Acidemia core Metab-OA IVD 3712 DBS, C5 MS/MS 3-MCC 3-Methylcrotonyl-CoA Carboxylase Deficiency core Metab-OA MCCC1 56922 DBS, C5-OH MCCC2 64087 MS/MS HMG 3-Hydroxy-3-Methylglutaric Aciduria core Metab-OA HMGCL 3155 DBS, C5-OH MS/MS MCD Holocarboxylase Synthase deficiency core Metab-OA HLCS 3141 DBS, C5-OH BTD 686 MS/MS BKT Beta-Ketothiolase Deficiency core Metab-OA ACAT1 38 DBS, C5-OH MS/MS GA1 Glutaric Acidemia Type I core Metab-OA GCDH 2639 DBS, C5-DC MS/MS CUD Carnitine Uptake Defect/Carnitine Transport core Metab-FAO SLC22A5 6584 DBS, C0 Defect MS/MS MCAD Medium-chain Acyl-CoA Dehydrogenase core Metab-FAO ACADM 34 DBS, C8 Deficiency MS/MS VLCAD Very Long-chain Acyl-CoA Dehydrogenase core Metab-FAO ACADVL 37 DBS, C14:1 Deficiency MS/MS LCHAD Long-chain L-3 Hydroxyacyl-CoA core Metab-FAO HADHA 3030 DBS, C16-OH Dehydrogenase Deficiency HADHB 3032 MS/MS TFP Trifunctional Protein Deficiency core Metab-FAO HADHA 3030 DBS, C16-OH HADHB 3032 MS/MS ASA Argininosuccinic Aciduria core Metab-AA ASL 435 DBS, Citrulline MS/MS CIT Citrullinemia, Type I core Metab-AA ASS1 445 DBS, Citrulline BCKDHA 593 MS/MS BCKDHB 594 MSUD Maple Syrup Urine Disease core Metab-AA DBT 1629 DBS, Leucine, DLD 1738 MS/MS Isoleucine BCKDK 10295 PPM1K 152926 CBS 875 HCY Homocystinuria core Metab-AA MTHFR 4524 DBS, Methionine MTR 4548 MS/MS MTRR 4552 PKU Classical Phenylketonuria core Metab-AA PAH 5053 DBS, Phenylalanine MS/MS TYR-I Tyrosinemia, Type I core Metab-AA FAH 2184 DBS, Tyrosine, MS/MS Succinylacetone BIOT Biotinidase Deficiency core Metab-other BTD 686 DBS, Biotidinase enzyme CF Cystic Fibrosis core Other- CFTR 1080 DBS, IRT IRT disorder GALT Classical Galactosemia core Metab-other GALT 2592 DBS, Galactose, GALT enzyme activity Cbl-C, D Methylmalonic acidemia with homocystinuria secondary Metab-OA MMACHC 25974 DBS, C3 MMADHC 27249 MS/MS MAL Malonic acidemia secondary Metab-OA MLYCD 23417 DBS, C3-DC MS/MS IBG/IBD Isobutyrylglycinuria secondary Metab-OA ACAD8 27034 DBS, C4 MS/MS 2MBG 2-Methylbutyrylglycinuria secondary Metab-OA ACADSB 36 DBS, C5 MS/MS 3MGA 3-Methylglutaconic aciduria secondary Metab-OA AUH 549 DBS, C5OH OPA3 80207 MS/MS TAZ 131118 2M3HBA 2-Methyl-3-hydroxybutyric aciduria secondary Metab-OA HSD17B10 3028 DBS, C5OH MS/MS SCAD Short-chain acyl-CoA dehydrogenase deficiency secondary Metab-FAO ACADS 35 DBS, C4 MS/MS M/SCHAD Medium/short-chain L-3-hydroxyacyl-CoA secondary Metab-FAO HADH 3033 DBS, C4OH dehydrogenase deficiency MS/MS GA-II Glutaric acidemia type II secondary Metab-FOA ETFA 2108 DBS, C4 ETFB 2109 MS/MS ETFDH 2110 MCAT Medium-chain ketoacyl-CoA thiolase deficiency secondary Metab-FAO HADHA 3030 DBS, C16OH HADHB 3032 MS/MS CPT-IA Carnitine palmitoyltransferase type I deficiency secondary Metab-FAO CPT1A 1374 DBS, CO/C16 + 18 MS/MS CPT-II Carnitine palmitoyltransferase type II deficiency secondary Metab-FAO CPT2 1376 DBS, C16 MS/MS CACT Carnitine acylcarnitine translocase deficiency secondary Metab-FAO SLC25A20 788 DBS, C16 MS/MS ARG Argininemia secondary Metab-AA ARG1 383 DBS, Arginine MS/MS CIT-II Citrullinemia, type II secondary Metab-AA SLC25A13 10165 DBS, Citrulline MS/MS MET Hypermethioninemia secondary Metab-AA MAT1A 4143 DBS, Methionine AHCY 191 MS/MS GNMT 27232 H-PHE Benign hyperphenylalaninemia secondary Metab-AA PAH 5053 DBS, Phenylalanine MS/MS BIOPT(BS) Biopterin defect in cofactor biosynthesis secondary Metab-AA GCH1 2643 DBS, Phenylalanine PTS 5805 MS/MS BIOPT(REG) Biopterin defect in cofactor regeneration secondary Metab-AA QDPR 5860 DBS, Phenylalanine PCBD1 5092 MS/MS TYR-II Tyrosinemia, type II secondary Metab-AA TAT 6898 DBS, Tyrosine MS/MS TYR-III Tyrosinemia, type III secondary Metab-AA HPD 3242 DBS, Tyrosine MS/MS GALE Galactoepimerase deficiency secondary Other- GALE 2582 DBS, Galactose disorder galactose GALK Galactokinase deficiency secondary Other- GALK1 2584 DBS, Galactose disorder galactose Additional RUSPseq conditions (Not on the RUSP)* (OTC) Ornithine transcarbamylase deficiency — Metab-AA OTC 5009 DBS, Citrulline. Screened MS/MS in CA since 2010. (CPS) Carbamoyl-phosphate synthetase deficiency — Metab-AA CPS1 1373 — No NBS. Included due to Hyperammonemia. (NAGSD) N-acetylglutamate synthase deficiency — Metab-AA NAGS 162417 — No NBS. Included due to Hyperammonemia. (Cbl-F) Methylmalonic aciduria and homocystinuria, — Metab-OA LMBRD1 55788 DBS, Related to Cbl-A, B Cbl-F type MS/MS and Cbl-C, D. (CMAMMA) Combined malonic and methylmalonic aciduria — Metab-OA ACSF3 197322 DBS, No NBS. Included MS/MS due to MMA. (MCEE) Methylmalonyl-CoA epimerase deficiency — Metab-OA MCEE 84693 DBS, No NBS. Included MS/MS due to MMA. (NKH) Nonketotic hyperglycemia — Metab-AA AMT 275 — No NBS. Included GLDC 2731 — due to DLD (PDH complex). *Reference: www.hrsa.gov/advisory-committees/heritable-disorders/rusp/index.html

TABLE 2 Target and Primer SEQ ID NOs Target Forward Reverse Sequence Primer Primer SEQ ID SEQ ID SEQ ID Target Name No No No ACAD8_exon_1-utr5_1 1 940 1879 ACAD8_exon_2-utr5_1 2 941 1880 ACAD8_exon_4-utr3_4d 3 942 1881 ACAD8_exon_5-utr3_6a 4 943 1882 ACAD8_exon_5-utr3_6b 5 944 1883 ACAD8_exon_5-utr3_6c 6 945 1884 ACAD8_exon_5-utr3_6f 7 946 1885 ACAD8_exon_6-utr3_1 8 947 1886 ACAD8_exon_7-utr3_1 9 948 1887 ACAD8_exon_8-utr3_19a 10 949 1888 ACAD8_exon_8-utr3_19d 11 950 1889 ACAD8_exon_8-utr3_19e 12 951 1890 ACAD8_exon_8-utr3_19i 13 952 1891 ACAD8_exon_9-utr3_3a 14 953 1892 ACADM_exon_10-utr3_2a 15 954 1893 ACADM_exon_10-utr3_2b 16 955 1894 ACADM_exon_11-utr3_3a 17 956 1895 ACADM_exon_1-utr5_2b 18 957 1896 ACADM_exon_2_1 19 958 1897 ACADM_exon_3_1 20 959 1898 ACADM_exon_4-utr5_3a 21 960 1899 ACADM_exon_4-utr5_3b 22 961 1900 ACADM_exon_4-utr5_3c 23 962 1901 ACADM_exon_5-utr3_1 24 963 1902 ACADM_exon_6-utr5_1 25 964 1903 ACADM_exon_7-utr3_2a 26 965 1904 ACADM_exon_7-utr3_3c 27 966 1905 ACADM_exon_8-utr3_1 28 967 1906 ACADS_exon_10-utr3_2a 29 968 1907 ACADS_exon_1-utr5_1 30 969 1908 ACADS_exon_2_1 31 970 1909 ACADS_exon_3_3a 32 971 1910 ACADS_exon_3_3b 33 972 1911 ACADS_exon_3_3c 34 973 1912 ACADS_exon_4_1 35 974 1913 ACADS_exon_5_1 36 975 1914 ACADS_exon_6_1 37 976 1915 ACADS_exon_7_1 38 977 1916 ACADS_exon_8_1 39 978 1917 ACADS_exon_9_1 40 979 1918 ACADSB_exon_10_1 41 980 1919 ACADSB_exon_11_2a 42 981 1920 ACADSB_exon_11_2b 43 982 1921 ACADSB_exon_12-utr3_37ab 44 983 1922 ACADSB_exon_1-utr5_1 45 984 1923 ACADSB_exon_2_1 46 985 1924 ACADSB_exon_3-utr5_1 47 986 1925 ACADSB_exon_4_1 48 987 1926 ACADSB_exon_5-utr5_1 49 988 1927 ACADSB_exon_6_1 50 989 1928 ACADSB_exon_7_1 51 990 1929 ACADSB_exon_8_1 52 991 1930 ACADSB_exon_9_1 53 992 1931 ACADVL_exon_1-utr5_1 54 993 1932 ACADVL_exon_2_1 55 994 1933 ACADVL_exon_3-utr5_4a 56 995 1934 ACADVL_exon_3-utr5_4b 57 996 1935 ACADVL_exon_3-utr5_4c 58 997 1936 ACADVL_exon_3-utr5_4d 59 998 1937 ACADVL_exon_4-utr3_2b 60 999 1938 ACADVL_exon_5-utr3_2a 61 1000 1939 ACADVL_exon_5-utr3_2b 62 1001 1940 ACADVL_exon_6-utr3_2a 63 1002 1941 ACADVL_exon_6-utr3_2b 64 1003 1942 ACADVL_exon_7-utr3_5a 65 1004 1943 ACADVL_exon_7-utr3_5b 66 1005 1944 ACADVL_exon_7-utr3_5c 67 1006 1945 ACADVL_exon_7-utr3_5d 68 1007 1946 ACAT1_exon_10-utr3_15a 69 1008 1947 ACAT1_exon_10-utr3_15b 70 1009 1948 ACAT1_exon_10-utr3_5e 71 1010 1949 ACAT1_exon_11_1 72 1011 1950 ACAT1_exon_12_1 73 1012 1951 ACAT1_exon_13-utr3_2a 74 1013 1952 ACAT1_exon_1-utr5_1 75 1014 1953 ACAT1_exon_2-utr5_1 76 1015 1954 ACAT1_exon_3-utr5_1 77 1016 1955 ACAT1_exon_4-utr5_1 78 1017 1956 ACAT1_exon_5_1 79 1018 1957 ACAT1_exon_6-utr3_3a 80 1019 1958 ACAT1_exon_7-utr3_5d 81 1020 1959 ACAT1_exon_8-utr3_1 82 1021 1960 ACAT1_exon_9-utr3_1 83 1022 1961 ACAT1_utr5_1 84 1023 1962 ACSF3_exon_10-utr3_21j 85 1024 1963 ACSF3_exon_10-utr3_61ay 86 1025 1964 ACSF3_exon_10-utr3_61bv 87 1026 1965 ACSF3_exon_10-utr3_61bx 88 1027 1966 ACSF3_exon_1-utr5_2a 89 1028 1967 ACSF3_exon_1-utr5_2b 90 1029 1968 ACSF3_exon_2-utr5_1 91 1030 1969 ACSF3_exon_3_1 92 1031 1970 ACSF3_exon_4_1 93 1032 1971 ACSF3_exon_5_1 94 1033 1972 ACSF3_exon_6-utr3_2a 95 1034 1973 ACSF3_exon_7-utr3_1 96 1035 1974 ACSF3_exon_8_1 97 1036 1975 ACSF3_exon_9-utr3_1 98 1037 1976 ACSF3_utr5_5_1 99 1038 1977 AHCY_exon_1-utr5_1 100 1039 1978 AHCY_exon_2-utr5_1 101 1040 1979 AHCY_exon_3_2a 102 1041 1980 AHCY_exon_4_1 103 1042 1981 AHCY_exon_5_1 104 1043 1982 AHCY_exon_6_1 105 1044 1983 AHCY_exon_7_1 106 1045 1984 AHCY_exon_8_1 107 1046 1985 AHCY_exon_9-utr3_3a 108 1047 1986 AMT_exon_1-utr5_4a 109 1048 1987 AMT_exon_1-utr5_4b 110 1049 1988 AMT_exon_1-utr5_4c 111 1050 1989 AMT_exon_1-utr5_4d 112 1051 1990 AMT_exon_2-utr3_6c 113 1052 1991 AMT_exon_2-utr3_6d 114 1053 1992 AMT_exon_2-utr3_6e 115 1054 1993 AMT_exon_2-utr3_6f 116 1055 1994 AMT_exon_3-utr3_1 117 1056 1995 AMT_exon_4-utr3_3a 118 1057 1996 AMT_exon_4-utr3_3b 119 1058 1997 ARG1_exon_1-utr5_1 120 1059 1998 ARG1_exon_2_1 121 1060 1999 ARG1_exon_3_2a 122 1061 2000 ARG1_exon_4_1 123 1062 2001 ARG1_exon_5_1 124 1063 2002 ARG1_exon_6_1 125 1064 2003 ARG1_exon_7_1 126 1065 2004 ARG1_exon_8-utr3_2a 127 1066 2005 ASL_exon_1_1 128 1067 2006 ASL_exon_10_1 129 1068 2007 ASL_exon_11_1 130 1069 2008 ASL_exon_12_1 131 1070 2009 ASL_exon_13-utr3_8b 132 1071 2010 ASL_exon_3_2a 133 1072 2011 ASL_exon_3_2b 134 1073 2012 ASL_exon_4_1 135 1074 2013 ASL_exon_6_1 136 1075 2014 ASL_exon_7_1 137 1076 2015 ASL_exon_8_1 138 1077 2016 ASL_exon_9_1 139 1078 2017 ASL_processed_transcript_1 140 1079 2018 ASL_utr5_1 141 1080 2019 ASS1_exon_10_1 142 1081 2020 ASS1_exon_11_1 143 1082 2021 ASS1_exon_12_1 144 1083 2022 ASS1_exon_13_1 145 1084 2023 ASS1_exon_14-utr3_1 146 1085 2024 ASS1_exon_1-utr5_1 147 1086 2025 ASS1_exon_2_1 148 1087 2026 ASS1_exon_3_1 149 1088 2027 ASS1_exon_4_1 150 1089 2028 ASS1_exon_5_1 151 1090 2029 ASS1_exon_6_1 152 1091 2030 ASS1_exon_7_1 153 1092 2031 ASS1_exon_8_2b 154 1093 2032 ASS1_exon_9_1 155 1094 2033 AUH_exon_10_1 156 1095 2034 AUH_exon_11-utr3_2a 157 1096 2035 AUH_exon_1-utr5_1 158 1097 2036 AUH_exon_3_1 159 1098 2037 AUH_exon_4_1 160 1099 2038 AUH_exon_5_1 161 1100 2039 AUH_exon_6_1 162 1101 2040 AUH_exon_8_1 163 1102 2041 AUH_exon_9_1 164 1103 2042 BCKDHA_exon_1_1 165 1104 2043 BCKDHA_exon_10-utr3_2a 166 1105 2044 BCKDHA_exon_10-utr3_2b 167 1106 2045 BCKDHA_exon_2-utr5_2b 168 1107 2046 BCKDHA_exon_3_1 169 1108 2047 BCKDHA_exon_4_1 170 1109 2048 BCKDHA_exon_5_1 171 1110 2049 BCKDHA_exon_6_1 172 1111 2050 BCKDHA_exon_7_1 173 1112 2051 BCKDHA_exon_8_1 174 1113 2052 BCKDHA_exon_9_1 175 1114 2053 BCKDHB_exon_10-utr3_1 176 1115 2054 BCKDHB_exon_11-utr3_8a 177 1116 2055 BCKDHB_exon_1-utr5_1 178 1117 2056 BCKDHB_exon_3_1 179 1118 2057 BCKDHB_exon_4_1 180 1119 2058 BCKDHB_exon_5_1 181 1120 2059 BCKDHB_exon_6-utr3_1 182 1121 2060 BCKDHB_exon_7-utr3_1 183 1122 2061 BCKDHB_exon_8-utr3_1 184 1123 2062 BCKDHB_exon_9-utr3_1 185 1124 2063 BCKDHB_processed_transcript_1_1 186 1125 2064 BCKDK_exon_1-utr5_2b 187 1126 2065 BCKDK_exon_2_1 188 1127 2066 BCKDK_exon_3_2b 189 1128 2067 BCKDK_exon_4_1 190 1129 2068 BCKDK_exon_5_3a 191 1130 2069 BCKDK_exon_5_3b 192 1131 2070 BCKDK_exon_5_3c 193 1132 2071 BCKDK_exon_6-utr3_1 194 1133 2072 BCKDK_exon_7-utr3_3a 195 1134 2073 BCKDK_exon_7-utr3_3b 196 1135 2074 BCKDK_exon_7-utr3_3c 197 1136 2075 BTD_exon_1-utr5_2b 198 1137 2076 BTD_exon_2-utr5_1 199 1138 2077 BTD_exon_3-utr5_1 200 1139 2078 BTD_exon_4_1 201 1140 2079 BTD_exon_5-utr3_4a 202 1141 2080 BTD_exon_5-utr3_4b 203 1142 2081 BTD_exon_5-utr3_4c 204 1143 2082 BTD_exon_5-utr3_4d 205 1144 2083 CBS_exon_10_2b 206 1145 2084 CBS_exon_11_1 207 1146 2085 CBS_exon_12_1 208 1147 2086 CBS_exon_13_1 209 1148 2087 CBS_exon_14-utr3_3a 210 1149 2088 CBS_exon_14-utr3_3b 211 1150 2089 CBS_exon_15-utr3_2a 212 1151 2090 CBS_exon_1-utr5_1 213 1152 2091 CBS_exon_2_1 214 1153 2092 CBS_exon_3_1 215 1154 2093 CBS_exon_4_1 216 1155 2094 CBS_exon_5_1 217 1156 2095 CBS_exon_6_1 218 1157 2096 CBS_exon_7_1 219 1158 2097 CBS_exon_8_2a 220 1159 2098 CBS_exon_9_1 221 1160 2099 CBS_processed_transcript-retained_intron 222 1161 2100 CFTR_exon_1 223 1162 2101 CFTR_exon_10 224 1163 2102 CFTR_exon_11n 225 1164 2103 CFTR_exon_12n 226 1165 2104 CFTR_exon_13 227 1166 2105 CFTR_exon_14_3a 228 1167 2106 CFTR_exon_14_3b 229 1168 2107 CFTR_exon_14_3c 230 1169 2108 CFTR_exon_15 231 1170 2109 CFTR_exon_16 232 1171 2110 CFTR_exon_17 233 1172 2111 CFTR_exon_18n 234 1173 2112 CFTR_exon_19n 235 1174 2113 CFTR_exon_20_2a1 236 1175 2114 CFTR_exon_20_2b1 237 1176 2115 CFTR_exon_21 238 1177 2116 CFTR_exon_22_2a 239 1178 2117 CFTR_exon_22_2b 240 1179 2118 CFTR_exon_23 241 1180 2119 CFTR_exon_24 242 1181 2120 CFTR_exon_25 243 1182 2121 CFTR_exon_26 244 1183 2122 CFTR_exon_27_6a 245 1184 2123 CFTR_exon_27_6b 246 1185 2124 CFTR_exon_27_6c 247 1186 2125 CFTR_exon_27_6d1 248 1187 2126 CFTR_exon_27_6e1 249 1188 2127 CFTR_exon_27_6f 250 1189 2128 CFTR_exon_2n1 251 1190 2129 CFTR_exon_3 252 1191 2130 CFTR_exon_4 253 1192 2131 CFTR_exon_5 254 1193 2132 CFTR_exon_6n 255 1194 2133 CFTR_exon_7 256 1195 2134 CFTR_exon_8 257 1196 2135 CFTR_exon_9 258 1197 2136 CFTR_intron_12 259 1198 2137 CFTR_intron_target_27b 260 1199 2138 CFTR_processed_transcript_3 261 1200 2139 CFTR_promotor 262 1201 2140 CFTR_rs139688774 263 1202 2141 CFTR_utr5_1 264 1203 2142 CFTR_utr5_2 265 1204 2143 CPS1_exon_10_1 266 1205 2144 CPS1_exon_11_1 267 1206 2145 CPS1_exon_12-utr5_1 268 1207 2146 CPS1_exon_13-utr5_1 269 1208 2147 CPS1_exon_14_1 270 1209 2148 CPS1_exon_15_1 271 1210 2149 CPS1_exon_16_1 272 1211 2150 CPS1_exon_17_1 273 1212 2151 CPS1_exon_18_1 274 1213 2152 CPS1_exon_19_1 275 1214 2153 CPS1_exon_1-utr5_1 276 1215 2154 CPS1_exon_2_1 277 1216 2155 CPS1_exon_20_1 278 1217 2156 CPS1_exon_21_1 279 1218 2157 CPS1_exon_22_1 280 1219 2158 CPS1_exon_23_1 281 1220 2159 CPS1_exon_24_1 282 1221 2160 CPS1_exon_25_1 283 1222 2161 CPS1_exon_26_1 284 1223 2162 CPS1_exon_27_1 285 1224 2163 CPS1_exon_28_1 286 1225 2164 CPS1_exon_29_1 287 1226 2165 CPS1_exon_3_1 288 1227 2166 CPS1_exon_30_1 289 1228 2167 CPS1_exon_31_1 290 1229 2168 CPS1_exon_32_1 291 1230 2169 CPS1_exon_33_1 292 1231 2170 CPS1_exon_34_1 293 1232 2171 CPS1_exon_35_16p 294 1233 2172 CPS1_exon_36_1 295 1234 2173 CPS1_exon_37_1 296 1235 2174 CPS1_exon_38-utr3_4a 297 1236 2175 CPS1_exon_4_1 298 1237 2176 CPS1_exon_5_1 299 1238 2177 CPS1_exon_6_1 300 1239 2178 CPS1_exon_7_1 301 1240 2179 CPS1_exon_8_1 302 1241 2180 CPS1_exon_9_1 303 1242 2181 CPS1_utr5_1_1 304 1243 2182 CPT1A_exon_10_1 305 1244 2183 CPT1A_exon_11_1 306 1245 2184 CPT1A_exon_12_1 307 1246 2185 CPT1A_exon_13_1 308 1247 2186 CPT1A_exon_14_1 309 1248 2187 CPT1A_exon_15_1 310 1249 2188 CPT1A_exon_16_1 311 1250 2189 CPT1A_exon_17_1 312 1251 2190 CPT1A_exon_19-utr3_8a 313 1252 2191 CPT1A_exon_1-utr5_1 314 1253 2192 CPT1A_exon_2_1 315 1254 2193 CPT1A_exon_20-utr3_1 316 1255 2194 CPT1A_exon_3_1 317 1256 2195 CPT1A_exon_4_1 318 1257 2196 CPT1A_exon_5-utr5_1 319 1258 2197 CPT1A_exon_6-utr5_1 320 1259 2198 CPT1A_exon_8_1 321 1260 2199 CPT1A_exon_9-utr3_1 322 1261 2200 CPT2_exon_1-utr5_2b 323 1262 2201 CPT2_exon_2_1 324 1263 2202 CPT2_exon_3_2a 325 1264 2203 CPT2_exon_4_4a 326 1265 2204 CPT2_exon_4_4b 327 1266 2205 CPT2_exon_4_4c 328 1267 2206 CPT2_exon_4_4d 329 1268 2207 CPT2_exon_5-utr3_3a 330 1269 2208 DBT_exon_10_1 331 1270 2209 DBT_exon_11-utr3_1 332 1271 2210 DBT_exon_11-utr3_71ac 333 1272 2211 DBT_exon_1-utr5_1 334 1273 2212 DBT_exon_2_1 335 1274 2213 DBT_exon_3_1 336 1275 2214 DBT_exon_4_1 337 1276 2215 DBT_exon_5_1 338 1277 2216 DBT_exon_6_1 339 1278 2217 DBT_exon_7_1 340 1279 2218 DBT_exon_8-utr3_2a 341 1280 2219 DBT_exon_8-utr3_2b 342 1281 2220 DBT_exon_9_1 343 1282 2221 DLD_exon_10-utr3_1 344 1283 2222 DLD_exon_11-utr3_1 345 1284 2223 DLD_exon_12-utr3_1 346 1285 2224 DLD_exon_13-utr3_19b 347 1286 2225 DLD_exon_1-utr5_1 348 1287 2226 DLD_exon_2-utr5_1 349 1288 2227 DLD_exon_3_1 350 1289 2228 DLD_exon_4_1 351 1290 2229 DLD_exon_5-utr5_1 352 1291 2230 DLD_exon_6-utr3_2a 353 1292 2231 DLD_exon_6-utr3_2b 354 1293 2232 DLD_exon_7-utr3_1 355 1294 2233 DLD_exon_8-utr3_1 356 1295 2234 DLD_exon_9-utr3_1 357 1296 2235 DLD_processed_transcript_1 358 1297 2236 DLD_utr3_1_2b 359 1298 2237 ETFA_exon_10-utr3_1 360 1299 2238 ETFA_exon_11-utr3_3a 361 1300 2239 ETFA_exon_12-utr3_5e 362 1301 2240 ETFA_exon_13-utr3_1 363 1302 2241 ETFA_exon_14-utr3_4a 364 1303 2242 ETFA_exon_1-utr5_1 365 1304 2243 ETFA_exon_2-utr3_1 366 1305 2244 ETFA_exon_3-utr5_1 367 1306 2245 ETFA_exon_4-utr5_1 368 1307 2246 ETFA_exon_5-utr5_1 369 1308 2247 ETFA_exon_6-utr5_1 370 1309 2248 ETFA_exon_9-utr5_1 371 1310 2249 ETFB_exon_1-utr5_1 372 1311 2250 ETFB_exon_2-utr5_8g 373 1312 2251 ETFB_exon_2-utr5_8h 374 1313 2252 ETFB_exon_3_1 375 1314 2253 ETFB_exon_5_8e 376 1315 2254 ETFB_exon_6-utr3_1 377 1316 2255 ETFDH_exon_10_1 378 1317 2256 ETFDH_exon_11_1 379 1318 2257 ETFDH_exon_12_1 380 1319 2258 ETFDH_exon_13-utr3_4a 381 1320 2259 ETFDH_exon_1-utr5_1 382 1321 2260 ETFDH_exon_2_1 383 1322 2261 ETFDH_exon_3_2a 384 1323 2262 ETFDH_exon_3_5e 385 1324 2263 ETFDH_exon_4-utr5_1 386 1325 2264 ETFDH_exon_5-utr5_1 387 1326 2265 ETFDH_exon_6_1 388 1327 2266 ETFDH_exon_7_1 389 1328 2267 ETFDH_exon_8_1 390 1329 2268 ETFDH_exon_9_1 391 1330 2269 FAH_exon_10_1 392 1331 2270 FAH_exon_11-utr3_1 393 1332 2271 FAH_exon_12-utr3_1 394 1333 2272 FAH_exon_13-utr3_3a 395 1334 2273 FAH_exon_1-utr5_6f 396 1335 2274 FAH_exon_2-utr5_1 397 1336 2275 FAH_exon_3_1 398 1337 2276 FAH_exon_4_4a 399 1338 2277 FAH_exon_5_1 400 1339 2278 FAH_exon_6_1 401 1340 2279 FAH_exon_7_1 402 1341 2280 FAH_exon_8_2a 403 1342 2281 FAH_exon_9_1 404 1343 2282 FAH_processed_transcript_1 405 1344 2283 FAH_utr5_2_1 406 1345 2284 GALE_exon_1_3a 407 1346 2285 GALE_exon_1_3b 408 1347 2286 GALE_exon_1_3c 409 1348 2287 GALE_exon_2_1 410 1349 2288 GALE_exon_4_1 411 1350 2289 GALE_exon_5-utr3_4a 412 1351 2290 GALE_exon_5-utr3_4b 413 1352 2291 GALE_exon_5-utr3_4c 414 1353 2292 GALE_exon_5-utr3_4d 415 1354 2293 GALK1_exon_1-utr-5_2b 416 1355 2294 GALK1_exon_2-utr3_4a 417 1356 2295 GALK1_exon_2-utr3_4b 418 1357 2296 GALK1_exon_2-utr3_4c 419 1358 2297 GALK1_exon_2-utr3_4d 420 1359 2298 GALK1_exon_3-utr3_2a 421 1360 2299 GALK1_exon_3-utr3_2b 422 1361 2300 GALK1_exon_4-utr3_1 423 1362 2301 GALT_exon_1-utr3_4a 424 1363 2302 GALT_exon_1-utr3_4b 425 1364 2303 GALT_exon_1-utr3_4c 426 1365 2304 GALT_exon_1-utr3_4d 427 1366 2305 GALT_exon_2-utr3_5a 428 1367 2306 GALT_exon_2-utr3_5b 429 1368 2307 GALT_exon_2-utr3_5c 430 1369 2308 GALT_exon_2-utr3_5d 431 1370 2309 GALT_exon_3-utr3_2a 432 1371 2310 GCDH_exon_10-utr3_1 433 1372 2311 GCDH_exon_11-utr3_2a 434 1373 2312 GCDH_exon_12-utr3_1 435 1374 2313 GCDH_exon_1-utr5_1 436 1375 2314 GCDH_exon_2-utr3_2a 437 1376 2315 GCDH_exon_3-utr3_4a 438 1377 2316 GCDH_exon_5_2a 439 1378 2317 GCDH_exon_6-utr3_1 440 1379 2318 GCDH_exon_7-utr3_1 441 1380 2319 GCDH_exon_8-utr3_1 442 1381 2320 GCDH_exon_9-utr3_2a 443 1382 2321 GCDH_exon_9-utr3_2b 444 1383 2322 GCH1_exon_1-utr5_2a 445 1384 2323 GCH1_exon_1-utr5_2b 446 1385 2324 GCH1_exon_2_1 447 1386 2325 GCH1_exon_3_1 448 1387 2326 GCH1_exon_4_1 449 1388 2327 GCH1_exon_5_1 450 1389 2328 GCH1_exon_6-utr3_6a 451 1390 2329 GCH1_exon_6-utr3_6b 452 1391 2330 GCH1_exon_6-utr3_6c 453 1392 2331 GCH1_exon_6-utr3_6d 454 1393 2332 GLDC_exon_10_1 455 1394 2333 GLDC_exon_11_1 456 1395 2334 GLDC_exon_12_1 457 1396 2335 GLDC_exon_13_1 458 1397 2336 GLDC_exon_14_1 459 1398 2337 GLDC_exon_15_1 460 1399 2338 GLDC_exon_16_1 461 1400 2339 GLDC_exon_17_2a 462 1401 2340 GLDC_exon_18_1 463 1402 2341 GLDC_exon_19_1 464 1403 2342 GLDC_exon_1-utr5_2a 465 1404 2343 GLDC_exon_1-utr5_2b 466 1405 2344 GLDC_exon_2_1 467 1406 2345 GLDC_exon_20_1 468 1407 2346 GLDC_exon_21_1 469 1408 2347 GLDC_exon_22_1 470 1409 2348 GLDC_exon_23_1 471 1410 2349 GLDC_exon_24_1 472 1411 2350 GLDC_exon_25-utr3_2a 473 1412 2351 GLDC_exon_3_1 474 1413 2352 GLDC_exon_4_1 475 1414 2353 GLDC_exon_5_1 476 1415 2354 GLDC_exon_6_1 477 1416 2355 GLDC_exon_7_1 478 1417 2356 GLDC_exon_8_1 479 1418 2357 GLDC_exon_9_1 480 1419 2358 GNMT_exon_1-utr5_1 481 1420 2359 GNMT_exon_2_1 482 1421 2360 GNMT_exon_3_1 483 1422 2361 GNMT_exon_4_1 484 1423 2362 GNMT_exon_5_1 485 1424 2363 GNMT_exon_6-utr3_1 486 1425 2364 HADH_exon_10-utr3_3a 487 1426 2365 HADH_exon_1-utr5_1 488 1427 2366 HADH_exon_2-utr5_1 489 1428 2367 HADH_exon_3_1 490 1429 2368 HADH_exon_4_1 491 1430 2369 HADH_exon_5_1 492 1431 2370 HADH_exon_6_1 493 1432 2371 HADH_exon_7_2a 494 1433 2372 HADH_exon_8_1 495 1434 2373 HADHA_exon_10-utr3_2a 496 1435 2374 HADHA_exon_11_1 497 1436 2375 HADHA_exon_12_1 498 1437 2376 HADHA_exon_13_1 499 1438 2377 HADHA_exon_14_1 500 1439 2378 HADHA_exon_15_1 501 1440 2379 HADHA_exon_16_1 502 1441 2380 HADHA_exon_17_1 503 1442 2381 HADHA_exon_18_1 504 1443 2382 HADHA_exon_19-utr3_3a 505 1444 2383 HADHA_exon_19-utr3_3b 506 1445 2384 HADHA_exon_1-utr5_1 507 1446 2385 HADHA_exon_2-utr5_1 508 1447 2386 HADHA_exon_4_1 509 1448 2387 HADHA_exon_5_1 510 1449 2388 HADHA_exon_6_1 511 1450 2389 HADHA_exon_7_1 512 1451 2390 HADHA_exon_8_1 513 1452 2391 HADHA_exon_9_1 514 1453 2392 HADHB_exon_10_1 515 1454 2393 HADHB_exon_12_1 516 1455 2394 HADHB_exon_13_1 517 1456 2395 HADHB_exon_14_1 518 1457 2396 HADHB_exon_15_1 519 1458 2397 HADHB_exon_16-utr3_2a 520 1459 2398 HADHB_exon_1-utr5_1 521 1460 2399 HADHB_exon_2-utr5_2a 522 1461 2400 HADHB_exon_3-utr5_3b 523 1462 2401 HADHB_exon_4_1 524 1463 2402 HADHB_exon_5_1 525 1464 2403 HADHB_exon_6_1 526 1465 2404 HADHB_exon_7_1 527 1466 2405 HADHB_exon_8_1 528 1467 2406 HADHB_exon_9_1 529 1468 2407 HLCS_exon_2_3a 530 1469 2408 HLCS_exon_2_3b 531 1470 2409 HLCS_exon_2_3c 532 1471 2410 HLCS_exon_3_1 533 1472 2411 HLCS_exon_4_2a 534 1473 2412 HLCS_exon_5_1 535 1474 2413 HLCS_exon_6_1 536 1475 2414 HLCS_exon_7_1 537 1476 2415 HLCS_exon_8_1 538 1477 2416 HLCS_exon_9-utr3_9a 539 1478 2417 HMGCL_exon_1-utr5_1 540 1479 2418 HMGCL_exon_2-utr3_1 541 1480 2419 HMGCL_exon_3-utr3_1 542 1481 2420 HMGCL_exon_4-utr3_2a 543 1482 2421 HMGCL_exon_5-utr3_2a 544 1483 2422 HMGCL_exon_6-utr3_4a 545 1484 2423 HMGCL_exon_7-utr3_1 546 1485 2424 HMGCL_exon_9-utr3_2a 547 1486 2425 HPD_exon_10_1 548 1487 2426 HPD_exon_11_1 549 1488 2427 HPD_exon_12_1 550 1489 2428 HPD_exon_13-utr3_1 551 1490 2429 HPD_exon_2-utr5_1 552 1491 2430 HPD_exon_3-utr5_2a 553 1492 2431 HPD_exon_3-utr5_2b 554 1493 2432 HPD_exon_4_1 555 1494 2433 HPD_exon_5_1 556 1495 2434 HPD_exon_6_1 557 1496 2435 HPD_exon_7_1 558 1497 2436 HPD_exon_8_1 559 1498 2437 HPD_exon_9_1 560 1499 2438 HPD_utr5_2_1 561 1500 2439 HSD17B10_exon_1-utr5_1 562 1501 2440 HSD17B10_exon_2_1 563 1502 2441 HSD17B10_exon_3_1 564 1503 2442 HSD17B10_exon_4_1 565 1504 2443 HSD17B10_exon_5_1 566 1505 2444 HSD17B10_exon_6-utr3_1 567 1506 2445 IVD_exon_10-utr3_1 568 1507 2446 IVD_exon_11-utr3_1 569 1508 2447 IVD_exon_12-utr3_1 570 1509 2448 IVD_exon_13-utr3_30aa 571 1510 2449 IVD_exon_13-utr3_9c 572 1511 2450 IVD_exon_14-utr3_1 573 1512 2451 IVD_exon_15_1 574 1513 2452 IVD_exon_16-utr3_2a 575 1514 2453 IVD_exon_1-utr5_2b 576 1515 2454 IVD_exon_2-utr5_1 577 1516 2455 IVD_exon_3_1 578 1517 2456 IVD_exon_4_1 579 1518 2457 IVD_exon_5_1 580 1519 2458 IVD_exon_6_1 581 1520 2459 IVD_exon_7_1 582 1521 2460 IVD_exon_8_1 583 1522 2461 IVD_exon_9_2b 584 1523 2462 LMBRD1_exon_10_1 585 1524 2463 LMBRD1_exon_11_1 586 1525 2464 LMBRD1_exon_12_1 587 1526 2465 LMBRD1_exon_13-utr3_1 588 1527 2466 LMBRD1_exon_14-utr3_1 589 1528 2467 LMBRD1_exon_15-utr3_1 590 1529 2468 LMBRD1_exon_16-utr3_2a 591 1530 2469 LMBRD1_exon_1-utr5_1 592 1531 2470 LMBRD1_exon_2-utr5_2a 593 1532 2471 LMBRD1_exon_2-utr5_2b 594 1533 2472 LMBRD1_exon_3_1 595 1534 2473 LMBRD1_exon_4_1 596 1535 2474 LMBRD1_exon_5_1 597 1536 2475 LMBRD1_exon_6_1 598 1537 2476 LMBRD1_exon_7_1 599 1538 2477 LMBRD1_exon_8_1 600 1539 2478 LMBRD1_exon_9_1 601 1540 2479 MAT1A_exon_1-utr5_1 602 1541 2480 MAT1A_exon_2_1 603 1542 2481 MAT1A_exon_3_1 604 1543 2482 MAT1A_exon_4_1 605 1544 2483 MAT1A_exon_5_1 606 1545 2484 MAT1A_exon_6_1 607 1546 2485 MAT1A_exon_7_a 608 1547 2486 MAT1A_exon_7_b 609 1548 2487 MAT1A_exon_8_1 610 1549 2488 MAT1A_exon_9-utr3_6a 611 1550 2489 MCCC1_exon_11-utr3_1 612 1551 2490 MCCC1_exon_12-utr3_1 613 1552 2491 MCCC1_exon_13-utr3_1 614 1553 2492 MCCC1_exon_14-utr3_1 615 1554 2493 MCCC1_exon_15-utr3_1 616 1555 2494 MCCC1_exon_16-utr3_1 617 1556 2495 MCCC1_exon_17-utr3_1 618 1557 2496 MCCC1_exon_18-utr3_1 619 1558 2497 MCCC1_exon_19-utr3_1 620 1559 2498 MCCC1_exon_1-utr5_1 621 1560 2499 MCCC1_exon_2-utr5_1 622 1561 2500 MCCC1_exon_3_1 623 1562 2501 MCCC1_exon_4-utr3_1 624 1563 2502 MCCC1_exon_5-utr5_1 625 1564 2503 MCCC1_exon_6-utr3_1 626 1565 2504 MCCC1_exon_7-utr3_1 627 1566 2505 MCCC1_exon_8-utr3_1 628 1567 2506 MCCC1_exon_9-utr3_1 629 1568 2507 MCCC2_exon_10_10a 630 1569 2508 MCCC2_exon_10_10f 631 1570 2509 MCCC2_exon_10_10h 632 1571 2510 MCCC2_exon_11-utr3_1 633 1572 2511 MCCC2_exon_12-utr3_1 634 1573 2512 MCCC2_exon_14-utr3_1 635 1574 2513 MCCC2_exon_15-utr3_1 636 1575 2514 MCCC2_exon_16-utr3_1 637 1576 2515 MCCC2_exon_17-utr3_5a 638 1577 2516 MCCC2_exon_1-utr5_1 639 1578 2517 MCCC2_exon_2_1 640 1579 2518 MCCC2_exon_3_1 641 1580 2519 MCCC2_exon_4_1 642 1581 2520 MCCC2_exon_5_2a 643 1582 2521 MCCC2_exon_6_1 644 1583 2522 MCCC2_exon_7_1 645 1584 2523 MCCC2_exon_8_1 646 1585 2524 MCCC2_exon_9_1 647 1586 2525 MCEE_exon_1-utr5_2a 648 1587 2526 MCEE_exon_2-utr5_1 649 1588 2527 MCEE_exon_3-utr3_2a 650 1589 2528 MCEE_exon_3-utr3_2b 651 1590 2529 MLYCD_exon_1-utr-5_2a 652 1591 2530 MLYCD_exon_1-utr5_2b 653 1592 2531 MLYCD_exon_2_1 654 1593 2532 MLYCD_exon_3_1 655 1594 2533 MLYCD_exon_4-utr3_12b 656 1595 2534 MLYCD_exon_4-utr3_12j 657 1596 2535 MLYCD_exon_4-utr3_12k 658 1597 2536 MMAA_exon_1-utr5_2a 659 1598 2537 MMAA_exon_1-utr5_2b 660 1599 2538 MMAA_exon_2-utr5_1 661 1600 2539 MMAA_exon_3-utr5_2a 662 1601 2540 MMAA_exon_4_1 663 1602 2541 MMAA_exon_5-utr5_1 664 1603 2542 MMAA_exon_6-utr3_13a 665 1604 2543 MMAB_exon_1-utr5_2b 666 1605 2544 MMAB_exon_2-utr5_1 667 1606 2545 MMAB_exon_3-utr5_1 668 1607 2546 MMAB_exon_4-utr3_1 669 1608 2547 MMAB_exon_5-utr3_1 670 1609 2548 MMAB_exon_6-utr3_1 671 1610 2549 MMAB_exon_7-utr3_1 672 1611 2550 MMAB_exon_8-utr3_1 673 1612 2551 MMAB_exon_9-utr3_9a 674 1613 2552 MMAB_utr3_2_1 675 1614 2553 MMACHC_exon_1-utr5_1 676 1615 2554 MMACHC_exon_2_1 677 1616 2555 MMACHC_exon_3_1 678 1617 2556 MMACHC_exon_4-utr3_21d 679 1618 2557 MMACHC_exon_4-utr3_6a 680 1619 2558 MMADHC_exon_1-utr5_2b 681 1620 2559 MMADHC_exon_2_1 682 1621 2560 MMADHC_exon_3_1 683 1622 2561 MMADHC_exon_4_1 684 1623 2562 MMADHC_exon_5_1 685 1624 2563 MMADHC_exon_6_1 686 1625 2564 MMADHC_exon_7_1 687 1626 2565 MMADHC_exon_8-utr3_2a 688 1627 2566 MMADHC_exon_8-utr3_2b 689 1628 2567 MTHFR_exon_10_1 690 1629 2568 MTHFR_exon_11-utr3_14a 691 1630 2569 MTHFR_exon_1-utr5_5d 692 1631 2570 MTHFR_exon_1-utr5_5e 693 1632 2571 MTHFR_exon_2_1 694 1633 2572 MTHFR_exon_3_1 695 1634 2573 MTHFR_exon_4_1 696 1635 2574 MTHFR_exon_5_1 697 1636 2575 MTHFR_exon_6_1 698 1637 2576 MTHFR_exon_7_1 699 1638 2577 MTHFR_exon_8_1 700 1639 2578 MTHFR_exon_9_1 701 1640 2579 MTR_exon_10_1 702 1641 2580 MTR_exon_11_1 703 1642 2581 MTR_exon_12_1 704 1643 2582 MTR_exon_13_1 705 1644 2583 MTR_exon_14_1 706 1645 2584 MTR_exon_15_1 707 1646 2585 MTR_exon_16_1 708 1647 2586 MTR_exon_17_1 709 1648 2587 MTR_exon_18_1 710 1649 2588 MTR_exon_19_1 711 1650 2589 MTR_exon_1-utr5_2a 712 1651 2590 MTR_exon_1-utr5_2b 713 1652 2591 MTR_exon_2_1 714 1653 2592 MTR_exon_20_1 715 1654 2593 MTR_exon_21_1 716 1655 2594 MTR_exon_22_1 717 1656 2595 MTR_exon_23_1 718 1657 2596 MTR_exon_24_1 719 1658 2597 MTR_exon_25_1 720 1659 2598 MTR_exon_26_1 721 1660 2599 MTR_exon_27_1 722 1661 2600 MTR_exon_28_1 723 1662 2601 MTR_exon_29_1 724 1663 2602 MTR_exon_3_1 725 1664 2603 MTR_exon_30_1 726 1665 2604 MTR_exon_31_3a 727 1666 2605 MTR_exon_31_3c 728 1667 2606 MTR_exon_32_1 729 1668 2607 MTR_exon_33-utr3_18a 730 1669 2608 MTR_exon_4_1 731 1670 2609 MTR_exon_5_1 732 1671 2610 MTR_exon_6-utr3_1 733 1672 2611 MTR_exon_7-utr3_1 734 1673 2612 MTR_exon_8-utr3_1 735 1674 2613 MTR_exon_9-utr3_1 736 1675 2614 MTRR_exon_10_1 737 1676 2615 MTRR_exon_11-utr3_2a 738 1677 2616 MTRR_exon_11-utr3_2b 739 1678 2617 MTRR_exon_12-utr3_1 740 1679 2618 MTRR_exon_13_2b 741 1680 2619 MTRR_exon_14-utr3_1 742 1681 2620 MTRR_exon_15-utr3_4a 743 1682 2621 MTRR_exon_1-utr5_1 744 1683 2622 MTRR_exon_2-utr5_1 745 1684 2623 MTRR_exon_3-utr3_1 746 1685 2624 MTRR_exon_4-utr3_1 747 1686 2625 MTRR_exon_5-utr3_1 748 1687 2626 MTRR_exon_6-utr3_1 749 1688 2627 MTRR_exon_7-utr3_1 750 1689 2628 MTRR_exon_8-utr3_1 751 1690 2629 MTRR_exon_9-utr3_1 752 1691 2630 MTRR_utr5_1_2a 753 1692 2631 MUT_exon_10_1 754 1693 2632 MUT_exon_11_1 755 1694 2633 MUT_exon_12-utr3_5a 756 1695 2634 MUT_exon_1-utr5_2a 757 1696 2635 MUT_exon_1-utr5_2b 758 1697 2636 MUT_exon_2_1 759 1698 2637 MUT_exon_3_1 760 1699 2638 MUT_exon_4_1 761 1700 2639 MUT_exon_5_1 762 1701 2640 MUT_exon_6_1 763 1702 2641 MUT_exon_7_1 764 1703 2642 MUT_exon_8_1 765 1704 2643 MUT_exon_9_1 766 1705 2644 NAGS_exon_1-utr5_2a 767 1706 2645 NAGS_exon_1-utr5_2b 768 1707 2646 NAGS_exon_2_1 769 1708 2647 NAGS_exon_3_1 770 1709 2648 NAGS_exon_4_3a 771 1710 2649 NAGS_exon_4_3c 772 1711 2650 NAGS_exon_5_1 773 1712 2651 NAGS_exon_6-utr3_2a 774 1713 2652 OPA3_exon_1-utr5_1 775 1714 2653 OPA3_exon_2-utr3_20a 776 1715 2654 OPA3_exon_2-utr3_20b 777 1716 2655 OPA3_exon_3-utr3_6a 778 1717 2656 OPA3_exon_3-utr3_6b 779 1718 2657 OTC_exon_10-utr3_2a 780 1719 2658 OTC_exon_1-utr5_1 781 1720 2659 OTC_exon_2_1 782 1721 2660 OTC_exon_3_1 783 1722 2661 OTC_exon_4_1 784 1723 2662 OTC_exon_5_1 785 1724 2663 OTC_exon_6_1 786 1725 2664 OTC_exon_7_1 787 1726 2665 OTC_exon_8_1 788 1727 2666 OTC_exon_9_1 789 1728 2667 PAH_exon_10_1 790 1729 2668 PAH_exon_11_1 791 1730 2669 PAH_exon_12_1 792 1731 2670 PAH_exon_13_1 793 1732 2671 PAH_exon_14-utr3_6a 794 1733 2672 PAH_exon_1-utr5_2b 795 1734 2673 PAH_exon_2-utr5_1 796 1735 2674 PAH_exon_3_1 797 1736 2675 PAH_exon_4_1 798 1737 2676 PAH_exon_5_1 799 1738 2677 PAH_exon_6_1 800 1739 2678 PAH_exon_7_2a 801 1740 2679 PAH_exon_8_3c 802 1741 2680 PAH_exon_9_2b 803 1742 2681 PCBD1_exon_1-utr5_1 804 1743 2682 PCBD1_exon_2_1 805 1744 2683 PCBD1_exon_3_1 806 1745 2684 PCBD1_exon_4-utr3_2a 807 1746 2685 PCCA_exon_10_1 808 1747 2686 PCCA_exon_11_1 809 1748 2687 PCCA_exon_12_1 810 1749 2688 PCCA_exon_13_1 811 1750 2689 PCCA_exon_14_1 812 1751 2690 PCCA_exon_15_1 813 1752 2691 PCCA_exon_16_1 814 1753 2692 PCCA_exon_17_1 815 1754 2693 PCCA_exon_18_1 816 1755 2694 PCCA_exon_19_1 817 1756 2695 PCCA_exon_1-utr5_1 818 1757 2696 PCCA_exon_2_1 819 1758 2697 PCCA_exon_20_1 820 1759 2698 PCCA_exon_21_1 821 1760 2699 PCCA_exon_22_1 822 1761 2700 PCCA_exon_23_1 823 1762 2701 PCCA_exon_24-utr3_1 824 1763 2702 PCCA_exon_25_1 825 1764 2703 PCCA_exon_26_1 826 1765 2704 PCCA_exon_27_1 827 1766 2705 PCCA_exon_28-utr3_1 828 1767 2706 PCCA_exon_3_1 829 1768 2707 PCCA_exon_4_1 830 1769 2708 PCCA_exon_5_1 831 1770 2709 PCCA_exon_6_1 832 1771 2710 PCCA_exon_7_1 833 1772 2711 PCCA_exon_8_1 834 1773 2712 PCCA_exon_9_1 835 1774 2713 PCCB_exon_10_1 836 1775 2714 PCCB_exon_11_1 837 1776 2715 PCCB_exon_12_1 838 1777 2716 PCCB_exon_13_1 839 1778 2717 PCCB_exon_14_1 840 1779 2718 PCCB_exon_15_2a 841 1780 2719 PCCB_exon_15_2b 842 1781 2720 PCCB_exon_16-utr3_1 843 1782 2721 PCCB_exon_17-utr3_1 844 1783 2722 PCCB_exon_18-utr3_1 845 1784 2723 PCCB_exon_19-utr3_12a 846 1785 2724 PCCB_exon_19-utr3_12c 847 1786 2725 PCCB_exon_1-utr5_1 848 1787 2726 PCCB_exon_2_1 849 1788 2727 PCCB_exon_4-utr5_1 850 1789 2728 PCCB_exon_5_1 851 1790 2729 PCCB_exon_6_1 852 1791 2730 PCCB_exon_7_1 853 1792 2731 PCCB_exon_8_1 854 1793 2732 PCCB_exon_9_1 855 1794 2733 PCCB_processed_transcript_1 856 1795 2734 PPM1K_exon_1-utr5_13d 857 1796 2735 PPM1K_exon_1-utr5_13e 858 1797 2736 PPM1K_exon_1-utr5_13g 859 1798 2737 PPM1K_exon_1-utr5_13h 860 1799 2738 PPM1K_exon_2_1 861 1800 2739 PPM1K_exon_3-utr5_1 862 1801 2740 PPM1K_exon_4_1 863 1802 2741 PPM1K_exon_5_1 864 1803 2742 PPM1K_exon_6-utr3_47aa 865 1804 2743 PPM1K_exon_6-utr3_47ac 866 1805 2744 PTS_exon_1-utr5_1 867 1806 2745 PTS_exon_2-utr5_3a 868 1807 2746 PTS_exon_3-utr5_1 869 1808 2747 PTS_exon_4-utr5_2a 870 1809 2748 PTS_exon_4-utr5_2b 871 1810 2749 PTS_exon_5-utr3_1 872 1811 2750 PTS_exon_6-utr3_2a 873 1812 2751 PTS_utr5_1 874 1813 2752 QDPR_exon_1-utr5_1 875 1814 2753 QDPR_exon_3-utr3_1 876 1815 2754 QDPR_exon_4-utr3_1 877 1816 2755 QDPR_exon_5_1 878 1817 2756 QDPR_exon_6-utr3_2a 879 1818 2757 QDPR_exon_7-utr3_3a 880 1819 2758 SLC22A5_exon_1-utr5_2a 881 1820 2759 SLC22A5_exon_1-utr5_2b 882 1821 2760 SLC22A5_exon_2_6f 883 1822 2761 SLC22A5_exon_3_15a 884 1823 2762 SLC22A5_exon_3_15o 885 1824 2763 SLC22A5_exon_4-utr3_1 886 1825 2764 SLC22A5_exon_5-utr3_1 887 1826 2765 SLC22A5_exon_6-utr3_6a 888 1827 2766 SLC22A5_exon_6-utr3_6f 889 1828 2767 SLC22A5_exon_7-utr3_1 890 1829 2768 SLC22A5_exon_8-utr3_1 891 1830 2769 SLC22A5_exon_9-utr3_4a 892 1831 2770 SLC25A13_exon_10_1 893 1832 2771 SLC25A13_exon_11_1 894 1833 2772 SLC25A13_exon_12_1 895 1834 2773 SLC25A13_exon_13_1 896 1835 2774 SLC25A13_exon_14_1 897 1836 2775 SLC25A13_exon_15_1 898 1837 2776 SLC25A13_exon_16_1 899 1838 2777 SLC25A13_exon_17-utr3_3a 900 1839 2778 SLC25A13_exon_1-utr5_1 901 1840 2779 SLC25A13_exon_2-utr5_1 902 1841 2780 SLC25A13_exon_3-utr5_1 903 1842 2781 SLC25A13_exon_4_1 904 1843 2782 SLC25A13_exon_5-utr3_1 905 1844 2783 SLC25A13_exon_6_1 906 1845 2784 SLC25A13_exon_7_1 907 1846 2785 SLC25A13_exon_8_1 908 1847 2786 SLC25A13_exon_9_1 909 1848 2787 SLC25A13_processed_transcript_2_1 910 1849 2788 SLC25A20_exon_1-utr5_1 911 1850 2789 SLC25A20_exon_2-utr5_1 912 1851 2790 SLC25A20_exon_3-utr3_1 913 1852 2791 SLC25A20_exon_4-utr3_1 914 1853 2792 SLC25A20_exon_5-utr3_1 915 1854 2793 SLC25A20_exon_6-utr3_1 916 1855 2794 SLC25A20_exon_7-utr3_1 917 1856 2795 SLC25A20_exon_8-utr3_1 918 1857 2796 SLC25A20_exon_9-utr3_3a 919 1858 2797 TAT_exon_1_4a 920 1859 2798 TAT_exon_1_4b 921 1860 2799 TAT_exon_2_1 922 1861 2800 TAT_exon_3_1 923 1862 2801 TAT_exon_4_1 924 1863 2802 TAT_exon_5_1 925 1864 2803 TAT_exon_6_1 926 1865 2804 TAT_exon_7_1 927 1866 2805 TAT_exon_8_1 928 1867 2806 TAT_exon_9-utr3_9a 929 1868 2807 TAT_exon_9-utr3_9b 930 1869 2808 TAZ_exon_1-utr5_2b 931 1870 2809 TAZ_exon_2_2a 932 1871 2810 TAZ_exon_3-utr3_3b 933 1872 2811 TAZ_exon_3-utr3_3c 934 1873 2812 TAZ_exon_5-utr3_5c 935 1874 2813 TAZ_exon_5-utr3_5d 936 1875 2814 TAZ_exon_5-utr3_5e 937 1876 2815 TAZ_exon_6-utr3_1 938 1877 2816 TAZ_exon_7-utr3_3a 939 1878 2817

TABLE 4 DNA Sequencing and Metabolic Data Analysis in 80 Newborns VUS in 8 MMA PMID for 8 P/LP in 64 PMID for 64 DBS ID Condition RUSPseq P/LP in 8 MMA genes genes MMA RUSPseq genes A1 mut⁰ MUT: P/LP MUT (c.C682T:p.R228X)^(#) 15643616, (c.C581T:p.P194L); LMBRD1 20631720, (c.G1464A:p.W488X); MMACHC 27060300, (c.A452G:p.H151R)^(#) 27233228 B1 mut⁰ MUT: P/LP, MMAA: MUT (c.T1620A:p.C540X)^(#), — 19862841, — P/P (c.C322T:p.R108C)^(#); MMAA 16281286, (c.G3A:p.M1I), 20301409 (c.931delA:p.K311fs) C1 mut⁰ MUT: P/P MUT (c.C982T:p.L328F*Hom)^(#) — 15643616, 25125334 D1 mut⁰ MUT: P/LP MUT — 15781192, (c.678_679insAATTTATG: 23045948, p.V227fs)^(#), (c.G607A:p.G203R)^(#); 10923046, LMBRD1 (c.G879A:p.W293X); 19375370, MMAB (c.137delC:p.P46fs) 17432548 E1 mut⁰ MUT: LP/LP MUT — 28973083 (c.C1843A:p.P615T), (c.C422T: p.A141V); MCEE (c. −245 − 1G > A) F1 mut⁰ MUT:P/VUS MUT (c.C682T:p.R228X)^(#) MUT (c.1846C > 15643616 T:p.R616C) G1 mut⁰ QC: low read depth — — — H1 mut⁰ MUT: P/P MUT (c.C693G:p.Y231X)^(#), — 27167370, c.1399C > T (p.R467X)^(#) 25736335, 23430940, 15643616, 12402345, 22727635 A2 mut⁰ MUT: P/P MUT (c.G607A:p.G203R*Hom)^(#); — 10923046, MMAB (c.T2A:p.M1K); MMADHC 19375370, (c.509delA:p.N170fs) 17432548 B2 mut⁰ False negative MUT (c.C454T:p.R152X)^(#) — ClinVar (MUT: P/-) C2 mut⁰ MUT: P/P MUT (c.1891delG:p.A631fs)^(#), — 16281286, PAH 9781015, (c.678_679insAATTTATG: 15781192, (c.G1243A: 7556322 p.V227fs)^(#); MMAB (c.135 − 2A > G) 23045948 p.D415N)^(#), (c.C498A: p.Y166X) D2 mut⁰ MUT: P/P MUT — 15643616, (c.2194_2197delGCCG/insAAGGT)^(#), 10923046 (c.C682T:p.R228X)^(#); MMAA (c.A829G:p.R277G) E2 mut⁰ MUT: LP/P MUT (c.C322T:p.R108C)^(#) , — 20301409, (c.C682T:p.R228X)^(#) 16281286, 15643616 F2 mut⁰ MUT: LP/LP MUT (c.G607A:p.G203R*Hom)^(#); — 10923046, MMACHC 19375370, (c.401_402de1:p.D134fs) 17432548 G2 mut⁰ MUT: P/P MUT (c.G1560C:p.K520N*Hom)^(#); — 17075691 MMAB (c.T426A:p.Y142X) H2 Cbl MMACHC: P/LP MMACHC — ClinVar C, D, F (c.326_329del:p.P109fs)^(#), (c.G482A:p.R161Q)^(#) A3 Cbl MMACHC: P/P MMACHC (c.271dupA:p.V90fs)^(#), — 19370762, C, D, F (c.C615G:p.Y205X)^(#) 20631720, 25687216, 20631720 B3 Cbl MMACHC: P/P MMACHC (c.82 − 2A > G), — 19370762, C, D, F (c.G609A:p.W203X)^(#) 16311595 C3 Cbl MMACHC: P/LP/P MMACHC (c.C28T:p.Q10X), — 19370762, C, D, F (c.T578C:p.L193P)^(#), 16311595 (c.G608A:p.W203X)^(#); MCEE (c.G428A:p.R143H); MUT (c.G1897A:p.V633I) D3 Cbl QC: failed DNA — — — C, D, F extraction E3 Cbl MMACHC: P/LP MMACHC (c.271dupA:p.V90fs)^(#), — 19370762, C, D, F (c.T578C:p.L193P)^(#) 20631720, 16311595 F3 Cbl False negative (-/-) — MMACHC — C, D, F (c.T176C:p.F59S) G3 Cbl MMACHC: P/P MMACHC — 19370762, C, D, F (c.271dupA:p.V90fs*Hom)^(#); MUT 20631720 (c.T2099C:p.M700T) H3 Cbl MMACHC: P/LP MMACHC — 19370762 C, D, F (c.326_329del:p.P109fs)^(#), (c.G482A:p.R161Q)^(#); MMAA (c.672delA:p.T224fs); MMADHC (c.G170A:p.W57X) A4 Cbl MMACHC: P/LP MMACHC — 19370762, C, D, F (c.567dupT:p.R189fs)^(#), 21835369 (c.G482A:p.R161Q)^(#) B4 Cbl MMACHC: P/P MMACHC — ClinVar PCCA C, D, F (c.326_329del:p.P109fs*Hom)^(#); (c.1070delG: MCEE (c.T305A:p.L102X); p.R357fs), MMAB (c.A653T:p.D218V); MUT (c.1963-2A>G) (c.C1849T:p.L617F) C4 Cbl MMACHC: P/P MMACHC — 27167370, MTHFR C, D, F (c.G608A:p.W203X*Hom)^(#) 19370762, (c.C778T:p.Q2 16311595 60X), (c.G160T:p.E5 4X) D4 Cbl MMACHC: P/P MMACHC (c.434dupT:p.I145fs), — 19370762, C, D, F (c.271dupA:p.V90fs)^(#) 20631720 E4 Cbl MMACHC: P/P MMACHC — 19370762, C, D, F (c.271dupA:p.V90fs*Hom)^(#) 20631720 F4 Cbl False negative MMADHC (c.680delC:p.P227fs) — — C, D, F (MMADHC: P/-) G4 Control — — — — H4 Control — — — — A5 Control — — — — B5 Control — MMADHC (c.T610C:p.F204L) — — C5 Control — — — — D5 Control — — — — E5 Control — — — — F5 Control — — — — G5 Control — MMAA (c.562 + 2T > C) — — H5 Control — MUT (c.C890T:p.T297I) — — A6 Control — MMAB (c.305delT:p.L102X) — — B6 Control — — — — C6 Control — — — — D6 Control — — — — E6 Control — MMACHC (c.259 − 2A > T) — — F6 Control — — — — G6 Control — — — — H6 Control — — — — PAH 26666653, (c.A204T: 23764561, p.R68S)^(#), 23500595 (c.A286T: p.K96X) (variants in cis) A7 Control — MMAA (c.C1114T:p.Q372X) — — B7 Control — — — — A8 MMA.FP — MMAA (c.G1079C:p.R360P) — — B8 MMA.FP — MMAB (c.644 + 1G > C) — MLYCD — (c.C886T: p.Q296X), (c.799 − 2A > T) (variants in cis) C8 MMA.FP — — — — D8 MMA.FP — — — — E8 MMA.FP — MUT (c.C1741T:p.R581X)^(#) — 27233228 F8 MMA.FP — — — — G8 MMA.FP — — — — H8 MMA.FP — — — — A9 MMA.FP — MMAA (c.A1072T:p.K358X) — — B9 MMA.FP — — — — C9 MMA.FP — — — — D9 MMA.FP — — — — E9 MMA.FP — LMBRD1 (c.C811T:p.Q271X) — — F9 MMA.FP — MUT (c.G278A:p.R93H)^(#) — 1670635 G9 MMA.FP — MMAB(c.585 − 1G > A)^(#) — 22695176 H9 MMA.FP — MUT (c.C1237T:p.Q413X) — — HPD (c.832 − 2A > T), (c.241 + 2T > C) A10 MMA.FP — MMAB (c.349 − 2A > T) — — B10 MMA.FP — MMACHC (c.463delG:p.G155fs) — — C10 MMA.FP — MCEE (c.85 − 2A > G) — — ACADVL (c.174delC: p.G58fs), (c.62 + 2T > C) D10 MMA.FP — — — — E10 MMA.FP LMBRD1: P/VUS LMBRD1 (c.C1321T:p.Q441X); LMBRD1 — FAH (variants in cis) MMAB (c.621delG:p.A207fs); (c.C1242T: (c.788delT: p.C414C) p.V263fs), (c.930delG: p.Q310fs) F10 MMA.FP — LMBRD1 (c.T2C:p.M1T); — — MMACHC (c.362delC:p.A121fs) G10 MMA.FP — LMBRD1 (c.G1464A:p.W488X) — — CPS1 (c.G741A: p.W247X), (c.C1891T: p.Q631X) H10 MMA.FP MUT: P/LP (variants MUT — — in cis) (c.1818delA:p.K606fs), (c.G1810A:p.V604I) A11 MMA.FP — — — — DBT (c.A31G: p.S11G), (c.889delA: p.I297fs) B11 MMA.FP — — — — C11 MMA.FP QC: failed DNA — — — extraction D11 MMA.FP — MMAA (c.534_535del:p.A178fs) — — GALT (c.569delG: p.W190fs) E11 MMA.FP — — — — NAGS(c.732delC: p.D244fs), (c.1399_1400i nsC:p.D467fs), (c.A1605G: p.X535W) F11 MMA.FP — — — Condition: mut⁰ = methylmalonyl-CoA mutase mut 0 enzymatic subtype; Cbl C, D, F = cobalamin deficieny type C, D and F. Control = healthy controls sample. FP = false positive case RUSPseq: Reportable finding with gene name and identified variants. P = pathogenic; LP = Likely pathogenic; VUS = variant of unknown significance; QC = quality control. P/LP in 8 MMA genes: Gene names in bold indicate genes identified with two P/LP in 1 gene. (^(#)) = known pathogenic variant in ClinVar or PubMed. VUS in 8 MMA genes: VUS are reported for TP and FP samples with only 1 P/LP in a MMA gene. P/LP in 64 RUSPseq genes: Variants identified in 64 genes other than the 8 MMA genes. (^(#)) = known pathogenic variant in ClinVar or PubMed. 

What is claimed is:
 1. A composition for performing PCR comprising: a universal primer; and a plurality of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer, wherein the forward and the reverse primer comprises a general 5′-A-B-3′ structure, where A represents the universal primer sequence and B represents a target specific sequence.
 2. The composition of claim 1, wherein the universal primer possesses a melting temperature of approximately 69° C. to approximately 72° C.
 3. The composition of claim 1, wherein the plurality of primer pairs is at least 50 primer pairs.
 4. The composition of claim 1, wherein the plurality of primer pairs is at least 500 primer pairs.
 5. The composition of claim 1, wherein the forward primers and reverse primers further comprise a spacer sequence, C, wherein the forward and reverse primers comprise a general 5′-A-C-B-3′ structure.
 6. The composition of claim 5, wherein the spacer in each forward primer consists of the sequence TCTG and the spacer in each reverse primer consists of the sequence AGAC.
 7. The composition of claim 1, wherein the universal primer and the plurality of primer pairs are at a ratio of 10:1.
 8. The composition of claim 1, wherein the universal primer sequence is SEQ ID:
 2818. 9. The composition of claim 1, wherein the target specific sequence for each forward primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 940-1878, and each reverse primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 1879-2817.
 10. A method of targeted sequencing of an individual, comprising the steps of: amplifying a plurality of target sequences in a sample using a first PCR reaction to create amplicons containing a universal primer sequence, wherein the first PCR reaction contains a universal primer, a plurality of forward primers, and a plurality of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer, wherein the forward and the reverse primer comprises a general 5′-A-B-3′ structure, where A represents the universal primer sequence and B represents a target specific sequence; generating a sequencing library from the amplicons using a second PCR reaction, wherein the second PCR reaction contains sequencing adapter primers comprising a general 5′-D-A-3′ structure, where D represents a sequencing adapter sequence and A represents the universal primer sequence; and sequencing the sequencing library on a sequencing platform.
 11. The method of claim 10, further comprising obtaining a sample.
 12. The method of claim 11, wherein the sample is dried blood spot.
 13. The method of claim 10, wherein the forward primers, reverse primers, and sequencing adapter primers further comprise a spacer sequence, C, wherein the forward and reverse primers comprise a general 5′-A-C-B-3′ structure and the sequencing adapter primers comprise a general 5′-D-A-C-3′ structure.
 14. The method of claim 10, wherein the universal primer possesses a melting temperature of approximately 69° C. to approximately 72° C.
 15. The method of claim 10, wherein the universal primer and the plurality of primer pairs are at a ratio of 10:1.
 16. The method of claim 10, wherein the universal primer sequence is SEQ ID:
 2818. 17. The method of claim 10, wherein the plurality of primer pairs is at least 50 primer pairs.
 18. The method of claim 10, wherein the plurality of primer pairs is at least 500 primer pairs.
 19. The method of claim 10, wherein the target specific sequence for each forward primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 940-1878, and each reverse primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 1879-2817.
 20. The method of claim 10, wherein the sequencing adapter sequence is selected from the group consisting of SEQ ID NOs: 2819-2820. 